Differentiation inhibitor

ABSTRACT

A novel human serrate-2 polypeptide consisting of a polypeptide containing the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing and having the effect of regulating the differentiation of undifferentiated cells involving stem cells; its gene; a process for producing the same; and an antibody specifically recognizing the polypeptide.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel bioactive substance which suppressesdifferentiation of undifferentiated cells.

2. Description of Related Art

Human blood and lymph contain various types of cells and each cell playsimportant roles. For example, erythrocytes carry oxygen; platelets havehemostatic action; and lymphocytes prevent infection. These variouscells originate from hematopoietic stem cells in the bone marrow.Recently, it has been clarified that the hematopoietic stem cells aredifferentiated to various blood cells, osteoclasts and mast cells bystimulation of various cytokines in vivo and environmental factors. Inthe cytokines, there have been found, for example, erythropoietin (EPO)for differentiation to erythrocytes; granulocyte colony stimulatingfactor(G-CSF) for differentiation to leukocytes; and platelet growthfactor (mpl ligand) for differentiation to megakaryocytes which areplatelet producing cells; and the former two examples have already beenclinically applied.

The differentiated blood cells are generally classified into two groupsconsisting of blood precursor cells which are destined to differentiateto specific blood series and hematopoietic stem cells which havedifferentiation ability to all series and self-replication activity. Theblood precursor cells can be identified by various colony assays;however, an identification method for the hematopoietic stem cells havenot been established. In these cells, stem cell factor (SCF),interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor(GM-CSF), interleukin-6 (IL-6), interleukin-1 (IL-1), granulocyte colonystimulating factor (G-CSF) and oncostatin M have been reported tostimulate cell differentiation and proliferation.

Trials for expansion of hematopoietic stem cells in vitro have beenconducted in order to replace bone marrow transplantation for applyinghematopoietic stem cell transplantation therapy or gene therapy.However, when the hematopoietic stem cells are cultured in the presenceof the above mentioned cytokines, multi-differentiation activities andself-replication activities, which are originally in the position of thehematopoietic stem cells, gradually disappeared and are changed to theblood cell precursors which only differentiate to specific series after5 weeks of cultivation, and multi-differentiation activity, which is oneof the specific features of the hematopoietic stem cells, is lost(Wanger et al. Blood 86, 512-523, 1995).

For proliferation of the blood precursor cells, a single cytokine is notsufficient, but rather the synergistic action of several cytokines isimportant. Consequently, in order to proliferate the hematopoietic stemcells while maintaining the specific features of the hematopoietic stemcells, it is necessary to add cytokines which suppress differentiationtogether with the cytokines which proliferate and differentiate theundifferentiated blood cells. In general, many cytokines which stimulateproliferation or differentiation of cells are known, but few cytokineswhich suppress cell differentiation are known. For example, leukemiainhibitory factor (LIF) has an action of proliferation of mouseembryonic stem cells without differentiation, but it has no actionagainst the hematopoietic stem cells or blood precursor cells.Transforming growthfactor (TGF-β) has suppressive action forproliferation against various cells, but has no fixed actions againstthe hematopoietic stem cells or blood precursor cells.

Not only blood cells but also undifferentiated cells, especially stemcells, are thought to be involved in tissue regeneration. Theseregeneration of tissues and proliferation of undifferentiated cells ineach tissue can be applied in various known ways (Katsutoshi Yoshizato,Regenration—a mechanism of regeneration, 1966, Yodosha Publ. Co.).

Notch is a receptor type membrane protein involved in regulation ofnerve cell differentiation found in Drosophia. Homologues of Notch arefound in various invertebrates and vertebrates including nematoda(Lin-12), Xenopus laevis (Xotch), mouse (Motch) and human (TAN-1).

Ligands of Notch in Drosophila are known. These are Drosophila Delta(Delta) and Drosophila Serrate (Serrate). Notch ligand homologues arefound in various animals similar to those of Notch receptors(Artavanis-Tsakonas et al., Science 268, 225-232, 1995).

Human Notch homologue, TAN-1 is found widely in the tissues in vivo(Ellisen et al., Cell 66, 649-661, 1991). Two Notch analogous moleculesother than TAN-1 have been reported (Artavanis-Tsakonas et al., Science268, 225-232, 1995). Expression of TAN-1 was also observed in CD34positive cells in blood cells by PCR (Polymerase Chain Reaction) (Milneret al., Blood 83, 2057-2062, 1994). However, in relation to humans, genecloning of human Delta and human Serrate, which are thought to be Notchligand, has not been reported.

In Drosophila Notch, binding with the ligand was studied andinvestigated in detail, and it was found that the Notch can be bound tothe ligand with Ca++ at the binding region, which is a repeated aminoacid sequence No. 11 and No. 12 in the amino acid sequence repeat ofEpidermal Growth Factor (EGF) (Fehon et al., Cell 61, 523-534, 1990,Rebay et al., ibid. 67, 687-699, 1991 and Japan. Patent PCT Unexam.Publ. 7-4 503123). EGF-like repeated sequences are conserved in Notchhomologues of other species. Consequently, the same mechanism in bindingwith ligand is assumed.

An amino acid sequence which is called DSL (Delta-Serrate-Lag-2) nearthe amino acid terminal, and EGF-like repeated sequence like in thereceptor are conserved in the ligand (Artavanis-Tsakonas et al., Science268, 225-232, 1995). EGF-like sequence has been found in thrombo-modulin(Jackman et al., Proc. Natl. Acad. Sci. USA 83, 8834-8838, 1986), lowdensity lipoprotein (LDL) receptor (Russell et al., Cell 37, 577-585,1984), and blood coagulating factor (Furie et al., Cell 53,505-518,1988), and is thought to play important roles in extracellularcoagulation and adhesion.

The vertebrate homologues of the cloned Drosophila Delta were found inchicken (C-Delta-1) and Xenopus laevis (X-Delta-1), and it has beenreported that X-Delta-1 had acted through Xotch in the generation of theprotoneuron (Henrique et al., Nature 375, 787-790, 1995 and Chitnis etal., ibid. 375, 761-766, 1995).

A vertebrate homologue of Drosophila Serrate was found in rat as ratJagged (Jagged)(Lindsell et al., Cell 80, 909-917, 1995). According toLindsell et al., mRNA of the rat Jagged is detected in the spinal cordof fetal rats. As a result of cocultivation of a myoblast cell line thatis forced to over express rat Notch with a rat Jagged expression cellline, suppression of differentiation of the myoblast cell line is found.However, the rat Jagged has no action against the myoblast cell linewithout forced expression of the rat Notch.

Considering the above reports, Notch and ligand thereof may be involvedin differentiation regulation of the nerve cells; however, except forsome myoblast cells, their actions against cells including blood cells,especially primary cells, are unknown.

As mentioned above, concerning undifferentiated cells, proliferationwhile maintaining their specificities has not been performed. Majorreasons are that factors suppressing differentiation of theundifferentiated cells have not been sufficiently identified.

SUMMARY AND OBJECTS OF THE INVENTION

A principal object of the present invention is to provide a compoundoriginated from novel factors which can suppress differentiation ofundifferentiated cells.

We have set up a hypothesis that the Notch and its ligand have an actionof differential regulation not only for neurogenic cells but also forvarious undifferentiated cells. However, in case of clinical applicationin humans, prior known different species such as chicken or Xenopuslaevos type Notch ligand have species-previously specific problems andanti-genicities. Consequently, to obtain prior unknown human Notchligand is essentially required. We had an idea that ligands of the humanNotch (TAN-1 etc.), which are a human Delta homologue (hereinafterdesignated as human Delta) and human Serrate homologue (hereinafterdesignated as human Serrate), may be found. Also we had an idea thatthese findings may be a candidate for drugs useful for differentialregulation of the undifferentiated cells. We have tried to discover thesame.

In order to discover human Notch ligands, we have analyzed amino acidsequences which are conserved in animals other than humans, and tried todiscover genes by PCR using mixed primers of the corresponding DNAsequence. As a result of extensive studies, we have succeeded inisolation of cDNAs coding amino acid sequences of two new molecules,novel human Delta-1 and novel human Serrate-1, and have prepared proteinexpression systems having various forms using these cDNAs. Also we haveestablished a purification method of the proteins which were purifiedand isolated, and already filed a patent application therefor(International Publication WO 97/19172).

Furthermore, we have tried to discover Drosophila Delta and Serrateanalogous molecules other than human Delta and human Serrate(hereinafter designated as human Delta-1 and human Serrate-1,respectively) of the above patent application in vertebrates.

We have tried to search on the data base of genetic sequences. Namely,based on the human Serrate-1 genetic sequence (amino acids sequence inSEQ ID NO: 5) which was at first discovered by us, we have found severalnumbers of gene fragments (length with 200-350 bp) with highhomologyfrom EST (Expressed Sequence Tag), which is a data of gene fragments ofrandom human cDNA sequence in the gene sequence data base Genank, usinga gene sequence search software Genetyx/CD (Software Development Co.).

These short length gene fragments were cloned by PCR, and these genefragments were used as probes to try cloning of the longer length genefragments from human fetal cDNA libraries. The thus isolated longer genefragments, of which the genetic sequences were determined, were againcompared with genetic sequence of human Serrate-1. As a result, a gene,which has relatively high homology with human Serrate-1, is identifiedand is designated as human Serrate-2. The full length of Serrate-2 genewas isolated successfully.

Furthermore, expression vectors of the said cloned Serrate-2 wereconstructed. A purification method of these proteins was established andthe said protein was purified and isolated. Antibodies against humanSerrate-2 are prepared using the said human Serrate-2, and apurification method of the said antibodies was established, then theactivity against undifferentiated blood cells was confirmed. The presentinvention was completed accordingly.

The present invention relates to a polypeptide comprising amino acidsequence of SEQ ID NO: 1, 2 or 3 of the sequence listing and thepolypeptide having differentiation suppressive action againstundifferentiated cells. Furthermore, the undifferentiated cells areundifferentiated cells except for those of the brain and nervous systemor muscular system cells, and in which the undifferentiated cells areundifferentiated blood cells. The present invention also relates topolypeptides having growth inhibitory action against vascularendothelial cells, a pharmaceutical composition containing the saidpolypeptides, a cell culture medium containing the said polypeptides anda cell culture medium in which the cells are undifferentiated bloodcells.

The present invention furthermore relates to a DNA coding a polypeptidecomprising amino acid sequence of SEQ ID NO: 1, 2 or 3 of the sequencelisting, the DNA having DNA sequence 90-731 DNA sequence 90-3254, or DNAsequence 90-3725 of SEQ ID NO: 4 of the sequence listing. The presentinvention still further relates to a recombinant DNA made by ligating aDNA coding a polypeptide comprising amino acid sequence of SEQ ID NO: 1,2 or 3 and a vector DNA which can express in the host cell and a cellcomprising transformed by the recombinant DNA.

The present invention also relates to a process for production ofpolypeptides by culturing cells and isolating the thus producedcompounds, and an antibody specifically recognizing the polypeptidehaving the amino acid sequence of SEQ ID NO. 1, 2 or 3 of the sequencinglisting.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is explained in detail in the following.

Preparation of cDNA necessary for gene manipulation, expression analysisby Northern blotting, screening by hybridization, preparation ofrecombinant DNA, determination of DNA base sequence and preparation ofcDNA library, all of which are series of molecular biologicalexperiments, can be performed according to a description of theconventional textbook for the experiments. The above conventionaltext-book of the experiments is, for example, Maniatis et al. ed.Molecular Cloning, A laboratory manual, 1989, Eds., Sambrook, J.,Fritsch, E. F. and Maniatis, T., Cold Spring Harbor Laboratory Press.

A novel compound of the present invention has at least polypeptides inthe sequence listing SEQ ID NO: 1-3. Mutants and alleles which naturallyoccur in nature are included in the polypeptide of the present inventionunless the polypeptides of the sequence listing, SEQ ID NO: 1, 2 or 3lose their properties. Modification and substitution of amino acids aredescribed in detail in the patent application by the name of Bennet etal. (National Unexam. Publ. WO 96/2645) and can be performed accordingto the description thereof. A modified polypeptide in the presentinvention means the modified polypeptide prepared by these amino acidreplacements and is defined as amino acid sequences having identity ofmore than 90% in its amino acid sequence.

A DNA sequence coding polypeptides of the sequence listing, SEQ ID NO:1-3 is shown in the sequence listing, SEQ ID NO: 4 as well as its aminoacid sequence. In these DNA sequences, even if an amino acid levelmutation is not generated, naturally isolated chromosomal DNA or cDNAthereof may have a possibility to mutate in the DNA base sequence as aresult of degeneracy of the genetic code without changing amino acidsequence coded by the DNA. A 5′-untranslated region and3′-untranslatedregion are not involved in amino acid sequencedetermination of the polypeptide, so the DNA sequences of these regionsare easily mutated. The base sequence obtained by these degeneracies ofthe genetic codes is included in the DNA of the present invention.

Undifferentiated cells in the present invention are defined as cellswhich can grow by specific stimulation, and cells which can bedifferentiated to cells having specific functions as a result ofspecific stimulations. These include undifferentiated cells of the skintissues, undifferentiated blood cells and nervous systems,undifferentiated cells of the muscular systems and undifferentiatedblood cells. These cells include those having self-replication activitywhich are called stem cells, and those having an ability to generate thecells of these lines. The differentiation-suppressive action meanssuppressive action for autonomous or heteronomous differentiation of theundifferentiated cells, and is an action for maintaining theundifferentiated condition. The brain and nervous undifferentiated cellscan be defined as cells having an ability to differentiate to the cellsof the brain or nerve having specific functions by specific stimulation.The undifferentiated cells of the muscular systems can be defined ascells having an ability to differentiate to the muscular cells havingspecific functions by specific stimulation. The blood undifferentiatedcells in the present invention can be defined as cell groups consistingof the blood precursor cursor cells which are differentiated to thespecific blood series identified by blood colony assay, andhematopoietic stem cells having differentiation to every series andself-replication activities.

In the sequence listing, the amino acid sequence in SEQ ID NO: 1 is asequence of the active center of the present invention of humanSerrate-2 minus the signal peptide, and corresponds to an amino acid No.1 to 217 in SEQ ID NO: 3 of the mature full length amino acid sequenceof human Serrate-2 of the present invention. The amino acid sequence inSEQ ID NO:2 is amino acid sequence of extracellular domain of thepresent invention of human Serrate-2 minus the signal peptide, andcorresponds to an amino acid No. 1 to 1058 in SEQ ID NO: 3 of the maturefull length amino acid sequence of human Serrate-2 of the presentinvention. The amino acid sequence of SEQ ID NO: 3 is the mature fulllength amino acid sequence of the human Serrate-2 of the presentinvention. The sequence of SEQ ID NOS: 4 and 5 is total amino acidsequence of human Serrate-2 of the present invention and cDNA coding thesame. The sequence of SEQ ID NOS: 6 and 7 is total amino acid sequenceof human Serrate-1 used in the present invention and cDNA coding thesame.

The left and right ends of the amino acid sequences in the sequencelistings indicate amino terminal (hereinafter designates as N-terminal)and carboxyl terminal (hereinafter designates as C-terminal),respectively, and the left and right ends of the nucleotide sequencesare 5′-terminal and 3′-terminal, respectively.

Cloning of human Notch ligand gene can be performed by the followingmethod. During the evolution of the organisms, a part of the amino acidsequences of the human Notch ligand is conserved. DNA sequencecorresponding to the conserved amino acid sequence is designed, and isused as a primer of RT-PCR (Reverse Transcription Polymerase ChainReaction), then a PCR template of human origin is amplituded by PCRreaction, whereby fragments of human Notch ligand can be obtained.Furthermore, RT-PCR primer is prepared by applying the known DNAsequence information of the Notch ligand homologue of the organismsother than humans, and the known gene fragments can be possibly obtainedfrom a PCR template of the said organisms.

In order to perform PCR for obtaining fragments of human Notch ligand,PCR for DSL sequence was considered, but a large number of combinationsof DNA sequence corresponding to amino acid sequence conserved in thisregion can be expected, and a design for PCR is difficult. As a result,PCR of the EGF-like sequence has to be selected. As explained above,since EGF-like sequence is conserved in a large number of molecules, toobtain the fragments and identification are extremely difficult. We havedesigned and prepared about 50 PCR primer sets, for example the primerset of the sequence shown in Referential example 1, and PCR wasperformed with these primer sets by using PCR template of cDNA preparedfrom poly A+RNA of various tissues of human origin, and more than 10 PCRproducts from each tissue were subcloned, as well as performingsequencing for more than 500 types. A clone having a desired sequence ofhuman Serrate-1 could be identified.

Namely, as shown in Reference example 1, the obtained PCR product iscloned in the cloning vector, transforming the host cells by usingrecombinant plasmid which contains the PCR product, culturing the hostcells containing the recombinant plasmid on a large scale, purifying andisolating the recombinant plasmid, checking the DNA sequence of PCRproduct which is inserted into the cloning vector, and trying to obtainthe gene fragment which may have a sequence of human Serrate-1 bycomparing with the sequence of the known Serrate homologue of otherspecies. We have succeeded in discovering a the gene fragment whichcontains a part of cDNA of human Serate-1, the same sequence of DNAsequence from 1272 to 1737 described in the sequence listing, SEQ ID NO:6. As shown in Referential example 2, using the thus obtained humanSerrate-1 gene fragment, full length cDNA is obtained from human cDNAlibrary. We have already filed a patent application with theseinventions (WO 97/19172).

In the present invention, there may be possibly to exist ligands otherthan this human Serrate-1 molecule, gene fragments showing high homologyin relation to the ligand to the gene sequence coding amino acidsequence of human Serrate-1 molecule, i.e. DNA sequence from 409 to 4062in the sequence listing SEQ ID NO: 6, are screened in the data base ofDNA sequence. Screening was performed by using gene sequence searchsoftware Genetyx/CD (software Development Co.) on the DNA/fragments ofrandom human cDNA sequence data base EST (Expressed Sequence Tag) ofGenbank (release 91, 1995).

Recently, DNA sequencing technology has progressed, and analysis oftotal geneomic DNA and full cDNA sequence of humans, nematoda,Arabidopsisthaliana Heynh, etc. were tried by random sequencing ofgenomic DNA and cDNA (Genome Directory, Nature, 377, 3S, 1995). In thehuman cDNA, EST project of TIGR (The Institute for Genomic Research),EST project of Washington Univ.—Merck, STS project of Colorado Univ.joined in these projects. Partial base sequences of cDNA provided bythese organizations are registered in DNA database of Genbank and EMBLand are disclosed. According to Genbank release 91 of October, 1995,cumulative registered numbers of EST clone are about 330,000 clones withaverage length 346 bp.

Based on data in these databases, gene sequences or amino acid sequencesof known or namely cloned novel molecules are searched by homologysearch. Possibility of existence of the analogues or similar familymolecules of these molecules can be known and the sequence informationof the partial DNA sequence can be obtained.

For analysis, commercially available analysis software can be used, oranalysis software attached to the database, for example BLAST of Genbankcan be used. By using these, analysis can be performed by accessing toNational Center of Biotechnology Information, U.S.A., Institute ofChemistry, Kyoto Univ., Japan, through WWW (world wide webb) or E-mail.

Gene sequence information of gene fragments (about 200-350 bp) with highsimilarity to the target gene can be obtained through these operations.The information of the obtained gene fragment includes general genesequence information together with clone name of the gene and organs ortissues in which the gene was extracted. In the information on DNAsequence, this is essentially raw data obtained by DNA sequence,including unknown DNA sequences with marked “n”, and incorrect DNAsequence information. Consequently, this DNA sequence information is notalways exact.

From this gene information, DNA sequences without unknown residues N arethought to be highly probable DNA sequence information. Further the mostprobable DNA sequences within these DNA sequences are compared and DNAfragments having significant homology are identified in this region (incase of a gene with 200 bases, similarity of DNA sequence above 40% ispreferable). The thus identified DNA fragment can be obtained from GenomSystem Inc., U.S.A. etc., if the name of the clone is known; however,because of knowing disclosed origin of organs, it can be also isolatedby PCR from cDNA of commercially available expression organs.

The thus found gene information is partial information, and unless totalinformation is obtained, a full length amino acid sequence, which may beencoded by the said partial sequence of gene, is not always analogoussimilar molecule used in the original homology search. Exact informationabout the molecule cannot be shown only by that information. As shownbelow, we have prepared a number of probes having homology and performedcloning by plaque hybridization; however, most DNA fragments did notcode the desired molecules. Consequently, this technique may betheoretically possible but is not easy technology.

We have prepared probes from about 50 DNA fragments which showedsimilarity with human Serrate-1 cDNA by PCR. Finally, as shown in thefollowing, cloning was performed by library screening technique. As aresult of determining the DNA sequence, a gene isolated by using 3 typesof DNA probes having sequence of SEQ ID NOS: 10, 11 and 12, which wereprepared based on DNA sequence clones registered in Genbank (Reg. No.T08853, R50026 and R46751) as described in Example 1, is a DNA fragmentcoding human Serrate-2 molecule.

Furthermore, using the thus isolated cDNA fragment as a probe andscreening a CDNA library of the expression organs, then the longer genehaving DNA sequence or full length cDNA gene can be screened. The fulllength cloning can be made by isotope labelling and non-isotopelabelling with the partial cloning gene, and screening the library byhybridization or other method. Isotope labelling can be performed by,for example, terminal labelling by using [32 p ] γ-ATP and T4polynucleotide kinase, or other labelling methods such as nicktranslation or primer extension method can be applied.

Furthermore, cloning of the full length gene or longer gene fragmentscan be performed by methods for extension of gene sequence with 5′-RACEor 3′-RACE method without using a library. In other methods, humanorigin cDNA library is ligated into the expression vector, expressing byCOS-7 or other cells, the ligated molecule is searched using receptorNotch protein and the objective gene is screened by expression cloningto isolate cDNA of the ligand. In the expression cloning, a cell sorterfractionation method which is applied with binding with polypeptidecontaining amino acid sequence of prior known 4 Notches such as TAN-1,and a detection method by film emulsion using radioisotope can bementioned.

In this specification, a method for obtaining genes of human Serrate-2is explained, and various methods clearly shown in this inventionincluding PCR, by which clonings of human Delta-1 and human Serrate-1were performed, can be applied for obtaining new Notch ligand familymolecules which have never been cloned. For example, the conserveddomains are found by comparison with amino acid sequence or DNA sequenceof human Serrate-1 or human Serrate-2, and cloning thereof is performedafter applying PCR, and also cloning can be performed by searching ESTbased on human Delta-1 or human Serrate-2. These cloned new Notch ligandfamily molecules can be used as the same human Serrate-2 shown in thepresent invention, for example by full length cloning, preparatingexpression vector, preparation of transformed cells, protein production,antibody production or screening the bioactive substances, anddifferentiation suppressive action for cells can be expected.

As shown in Example 2, these three gene fragments are labelled withradioisotope to prepare hybridization probes, screened using cDNA ofhuman fetal brain origin as the screening library, whereupon the DNAsequence of the thus obtained clones is determined, and found to behighly similar with human Serrate-1 in full length of DNA nucleotidesequence. In these screenings, a full length cDNA sequence encoding afull length amino acids sequence cannot be cloned. A further DNA probeis prepared based on the cloned DNA sequence, and again screening isperformed, but the full length gene cannot be identified. Finally, genecloning containing the translation initiation Met codon is performed by5′-RACE method, the DNA sequence is determined and finally we succeededin cloning of cDNA encoding full length of gene sequence of humanSerrate-2. The thus cloned cDNA was ligated as shown in Example 2 andcDNA encoding the full length of the said human Serrate-2 can beobtained.

Examples of plasmids integrated with cDNA are, for example, E. colioriginated pBR322, pUC18, pUC19, pUC118 and pUC119 (Takara Shuzo Co.,Japan), but other plasmids can be used if they replicate and proliferatein the host cells. Examples of phage vectors integrated with cDNA are,for example, λ gt10 and λ gt11, but other vectors can be used if theycan grow in the host cells. The thus obtained plasmids are transducedinto suitable host cells such as genus Escherichia and genus Bacillususing calcium chloride method. Examples of the above genus Escherichiaare Escherichia coli K12HB101, MC1061, LE392, JM109. Examples of theabove genus Bacillus is Bacillus subtilis MI114. Phage vector can beintroduced into the proliferated E. coli by the in vitro packagingmethod (Proc. Natl. Acad. Sci., 71: 2442, 1978).

The cloned full length DNA sequence was compared with the database(Genbank release 93, 1996), and it was found that the total sequence isa novel sequence, although there are partially the previously mentionedthree EST clones and several EST clone data as non-identical partialsequences other than those three EST clones.

Furthermore, the said amino acid sequence of human Serrate-2, i.e. aminoacid sequences in SEQ ID NO: 1, 2 and 3, were compared with the databaseof the prior known amino acid sequence (SWISS-PROT, release 32, 1995 andGenbank CDS, release 93, 1996), and found that there are no identicalamino acid sequences and that these are novel sequences. According to acomparison in amino acid sequence of human Serrate-1 and Serratehomologue of the other organisms, the homologies with human Serrate-1,Drosophila Serrate, and rat jagged are 53.1%, 34.3%, and 52.3%,respectively. The substance of the present invention is different fromthese substances and is a novel substance having new amino acidsequences and is first discovered by the present inventors.

The amino acid sequence was analyzed in hydrophilic part and hydrophobicpart according to a method by Kyte-Doolittle (J. Mol. Biol., 157: 105,1982). Results indicate that in the amino acid sequence listed in SEQ IDNO: 5, amino acid sequence of a precursor of full length gene consistsof 1238 amino acids residue from −26 to 1212 in amino acid sequence, andthe signal peptide domain is estimated to correspond amino acid sequenceof 26 amino acids residue from No. −26 methionine to No. −1 proline;extracellular domain: 1055 amino acids residue from No. 1 methionine toNo. 1055 glycine; transmembrane domain: 24 amino acids residue from No.1056 leucine to No. 1079 tryptophane; and intracellualr domain: regionfrom No. 1080 threonine to No. 1212 glutamate. These domains are theestimated domain construction from the amino acid sequence, and theactual form may differ from the above structure, and the constituentamino acids of each domain hereinabove defined may change 5 to 10 aminoacids per sequence.

In the amino terminal (N-terminal), as shown in Example 6,identification of N-terminal amino acid sequence of the purified ligandpolypeptides EXS2Fc and EXS2FLAG of the present invention was performedand found that it was methionine of No. 1 in SEQ ID NO: 1-3.Consequently, signal peptide is at least from −26 methionine to −1proline in SEQ ID NO: 5.

The family molecules of Notch ligand in relation to extracelluar domainhave evolutionally conserved common sequence, i.e. DSL sequence andrepeated EGF-like sequence. As a result of comparison with amino acidsequence of human Serrate-2 and human Serrate-1, the conserved sequenceis estimated from amino acid sequence Serrate-2. Namely, DSL sequencecorresponds to the 43 amino acid residue from No. 72 cysteine to No. 214cysteine of the amino acid sequence in the sequence listing, SEQ ID NO:5.

EGF-like sequence exists with 16 repeats wherein, in the amino acidsequence in the sequence listing, SEQ ID NO: 5, the first EGf-likesequence from No. 217 cysteine to No. 247 cysteine; the second EGF-likesequence from No. 250 cysteine to No. 278 cysteine; the third EGF-likesequence from No. 285 cysteine to No. 318 cysteine; the fourth EGF-likesequence from No. 325 cysteine to No. 356 cysteine; the fifth EGF-likesequence from No. 363 cysteine to No. 394 cysteine; the sixth EGF-likesequence from No. 401 cysteine to No. 432 cysteine; the seventh EGF-likesequence from No. 439 cysteine to No. 469 cysteine; the eighth EGF-likesequence from No. 476 cysteine to No. 507 cysteine; the ninth EGF-likesequence from No. 514 cysteine to No. 545 cysteine; the 10th EGF-likesequence from No. 563 cysteine to No. 607 cysteine; the 11th EGF-likesequence from No. 614 cysteine to No. 645 cysteine; the 12th EGF-likesequence from No. 652 cysteine to No. 683 cysteine; the 13th EGF-likesequence from No. 690 cysteine to No. 721 cysteine; the 14th EGF-likesequence from No. 729 cysteine to No. 760 cysteine; the 15th EGF-likesequence from No. 767 cysteine to No. 798 cysteine; and the 16thEGF-like sequence from No. 805 cysteine to No. 836 cysteine.

On these cysteine residues, there are 2 cysteine residues between the9th EGF-like sequence and the 10th EGF-like sequence. Also there are 6cysteine residues to the direction of N-terminal of DSL sequence and16cysteine residues to the direction of C-terminal in the 16th EGF-likesequence. These cysteine residues including EGF-like sequence areconserved in almost the same position of the human Serrate-1.

A part for sugar chain attachment is estimated from amino acid sequenceof the human Serrate-2 as No. 127, 544, 593, 726 and 1032 asparagineresidue in the sequence listing, SEQ ID NO: 3 as a possible binding siteof N-glycoside bonding for N-acetyl-D-glycosamine. O-glycoside bond ofN-acetyl-D-galactosamine is estimated to be a serine or threonineresidue rich part. Protein bound with sugar chain is generally thoughtto be stable in vivo and to have strong physiological activity.Consequently, in the amino acid sequence of polypeptide having sequenceof the sequence listing, SEQ ID NO: 1, 2 or 3, polypeptides havingN-glucoside or O-glucoside bond with sugar chain ofN-acetyl-D-glucosamine or N-acetyl-D-galactosamine is included in thepresent invention.

As a result of studies on binding of Drosophila Notch and its ligand,amino acid region necessary for binding with ligand of Drosophila Notchwith the Notch is from N-terminal to DSL sequence of the mature protein,in which signal peptide is removed (Japan. Pat. PCT Unexam. Publ. No.7-503121). Furthermore, as a result of the similar studies, a studyusing nematoda by Fitzgerald and Greenwald (Development, 121, 4275-4282,1995) clearly indicated that full length of Notch ligand-like moleculeAPX-1 from amino terminal to DSL domain was sufficient length foractivation of Notch-like receptor. These facts indicate that a domainnecessary for expression of ligand action of human Serrate-2 molecule isa novel amino acid sequence of the sequence listing, SEQ ID NO: 1.

Northern blotting can be performed by using DNA encoding a part or allof gene sequence in the sequence listing, SEQ ID NO: 4. Consequently, amethod for detection of expression of these genes can be achieved byapplying with hybridization or PCR by using complementary nucleic acidsof above 12 mer or above 16 mer, preferably above 18 mer having nucleicacid sequence of a part of sequence in the sequence listing SEQ ID NO:4, i.e. antisense DNA or antisense RNA, its methylated,methylphosphated, deaminated, or thiophosphated derivatives. By the samemethod, detection of homologues of the gene of other organisms such asmice or gene cloning can be achieved.

Further cloning of genes in the genome including humans can be made.Using these genes cloned by such like methods, further detailedfunctions of the human Serrate-2 of the present invention can beidentified. For examples using the modern gene manipulation techniques,every method including transgenic mouse, gene targeting mouse or doubleknockout mouse in which genes relating to the gene of the presentinvention are inactivated, can be applied. If abnormalities in thegenome of the present gene is found, application to gene diagnosis andgene therapy can be made.

As described in Example 3, an expression in normal human tissues isobserved in many tissues, and length of the expressed mRNA is one typeof the mRNA with about 5 kb length. This means that detecting theexpression of mRNA of the said molecule can be applied for diagnosis ordetection of malignant tumors in the part of normal organs in whichexpression of these mRNA cannot be observed. Furthermore, by referringto patterns of the expressed organs, use of human Serrate-2, for whichconcrete use is not indicated in the present invention, can be found.

A transformant in which vector pUCSR-2, which contains cDNA coding totalamino acid sequence of human Serrate-2 of the present invention, istransformed into E. coli JM109, has been deposited in the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, MITI, of 1-1-3, Higashi, Tsukuba-shi,Ibaragi-ken, Japan. as E. coli: JM109-pUCsr-2. Date of deposit was Oct.28, 1996, and deposition No. is FERM BP-5727.

Expression and purification of various forms of human Serrate-2 usingcDNA coding amino acid sequence of human Serrate-2 isolated by the abovemethods are known in the literature (Kriegler, Gene Transfer andExpression-A Laboratory Manual Stockton Press, 1990 and Yokota et al.Biomanual Series 4, Gene transfer and expression and analysis, YodoshaCo., 1994). A cDNA coding the amino acid sequence of the isolated saidhumanSerrate-2 is ligated to preferred expression vector and is producedin the host cells of eukaryotic cells such as animal cells and insectcells or prokaryotic cells such as bacteria.

In the expression of the molecule of the present invention, DNA encodinga polypeptide of the present invention may have the translationinitiation condon in 5′-terminal and translation termination codon in3′- terminal. These translation initiation codon and translationtermination codon can be added by using preferred synthetic DNA adapter.Furthermore, for expression of the said DNA, promoter is linked upstreamof the DNA sequence. Examples of vector are plasmid originated fromBacillus, plasmid originated from yeast or bacteriophage such as I-phageand animal virus such as retrovirus and vaccinia virus.

Examples of promoters used in the present invention are any promoterssuitable for corresponding to the host cells used in gene expression.

In case that the host cell in the transformation is genus Escherichia,tac-promoter, trp-promoter and lacpromoter are preferred, and in case ofhost of genus Bacillus, SPO1 promoter and SPO2 promoter are preferred,and in case of host of yeast, PGK promoter, GAP promoter and ADHpromoter are preferred.

In case that the host cell is animal cells, a promoter originated fromSV40, promoter of retrovirus, metallothionein promoter and heatshockpromoter can be applied.

Expression of the polypeptide of the present invention can be effectedby using only DNA encoding the amino acid sequence of the sequencelisting, SEQ ID NO: 1, 2 or 3. However, a protein having an additionalspecific function can be produced by using DNA, to which is added cDNAencoding the known antigen epitope for easier detection of the producedpolypeptide or to which is added cDNA encoding the immunoglobulin Fc forforming a multimer of the said human Serrate-2.

As shown in Example 4, we have prepared expression vectors, whichexpress extracellular proteins, as follow.

1) DNA encoding the amino acids from No. 1 to 1055 in amino acidsequence in the sequence listing, SEQ ID NO: 2.

2) DNA encoding chimera protein to which added polypeptide having 8amino acid, i.e. an amino acid sequence consisting of Asp Tyr Lys AspAsp Asp Asp Lys (hereinafter designates FLAG sequence, the sequencelisting, SEQ ID NO: 25), in the C-terminal of the amino acids from No. 1to 1055 in amino acid sequence in the sequence listing, SEQ ID NO: 2,and

3) DNA encoding chimera protein to which is added Fc sequence below thehinge region of human IgG1 (refer to International Patent Unexam. Publ.WO 94/02053) in the C-terminal of the amino acids from No. 1 to 1055 inamino acid sequence in the sequence listing, SEQ ID NO: 2, and to havedimer structure by disulfide bond in the hinge region, are ligatedindividually with the expression vector pMKITNeo (Maruyama et al., JapanMolecular Biology Soc. Meeting Preliminary lecture record, obtainablefrom Dr. Maruyama in Tokyo Medical and Dental College) to prepareextracellular expression vectors of human Serrate-1.

The expression vectors, which expresses full-length protein, can heprepared as follows.

4) DNA encoding amino acids from No. 1 to 1212 in the sequence listing,SEQ ID NO: 3 and

5) DNA encoding chimera protein to which is added polypeptide havingFLAG sequence in the C-terminal of amino acids from No. 1 to 1212 in thesequence listing, SEQ ID NO: 3 re ligated individually with theexpression vector PMKIT-Neo to prepare the full-length expression vectorof human Serrate-2. The transformation is prepared by using expressionplasmid containing DNA encoding the thus constructed said humanSerrate-2.

Examples of the host a re genus Escherichia, genus Bacillus, Yeast andanimal cells. Examples of animal cells are simian cell COS-7 and Vero,Chinese hamster cell CHO and silk worm cell SF9.

As shown in Example 5, the above 5 type expression vectors aretransduced individually; the human Serrate-2 is expressed in COS-7 cell(obtainable from the Institute of Physical and Chemical Research, CellDevelopment Bank, RCB0539); and the transformants which were transformedby these expression plasmids, can be obtained. Furthermore, humanSerrate-2 polypeptide can be produced by culturing the transformantsunder suitable culture conditions in medium by known culture methods.

The human Serrate-2 polypeptide can be isolated and purified from theabove cultured mass, in general, by the following methods.

For extraction of the substance from cultured microbial cells or cells,microbial cells or cells are collected by known method such ascentrifugation after the cultivation, suspended in siutable buffersolution, whereafter the microbial cells or cells are disrupted by meansof ultrasonication, lysozyme and/or freeze-thawing and a crude extractof human Serrate-2 protein is collected by centrifugation or filtration.The buffer solution may contain protein-denaturing agents such as ureaand guanidine hydrochloride or surface active agents such as TritonX-100. In case of secretion in the cultured solution, the cultured massis separated by known methods such as centrifugation to separate frommicrobial cells or cells, and the supernatant solution is collected.

The thus obtained human Serrate-2, which are contained in the cellextracts or cell supernatants, can be purified by known proteinpurification methods. During the purification process, for confirmationof existence of the protein, in case of the fused protein of the aboveFLAG and human IgGFc, it can be detected by immunoassay using antibodyagainst known antigen epitope and can be purified. In case the fusedprotein is not expressed as such, the antibody against human Serrate-2in Example 7 can be used for detection.

A more useful purification method is an affinity chromatography usingantibody. Antibodies used in this case are antibodies described inExample 7. For fused protein, antibodies against other than humanSerrate-2 are used, for example antibody against FLAG in the case ofFLAG, and protein G or protein A in the case of human IgGFc as shown inExample 6.

Physiological functions of the thus purified human Serrate-2 protein orhuman Serrate-2 can be identified by various assay methods, for example,physiological activity assaying using cell lines and animals such asmice and rats, assay methods of intracellular signal transduction basedon molecular biological means and binding with Notch receptor etc.

For that action, mainly an action suppressing cell differentiation willbe expected, and actions such as stimulating tissue regeneration, etc.can be expected.

Namely, we have found that, as shown in Example 8, in the umbilical cordblood derived blood undifferentiated cells in which CD34 positive cellfraction is concentrated, polypeptides of the present invention havesuppressive action of colony forming action against bloodundifferentiated cells, which shows colony formation in the presence ofcytokines.

Furthermore, as shown in Example 9, we have found that as a result ofadding IgG1 chimera protein of human Serrate-2 to the liquid culture inthe presence of cytokines, the human Serrate-2 had activities forsignificantly decreasing LTC-IC (Long-Term Culture-Initiating Cells)counts, which were positioned with most undifferentiated blood stemcells in the human blood undifferentiated cells.

These results indicate that the human Serrate-2 suppressesdifferentiation of blood undifferentiated cells, and these actionsspread from blood stem cells to colony forming cells. Furthermore,pharmaceuticals containing the polypeptide of the present invention haveaction for protection and release of the bone marrow suppressive action,which is observed in adverse effects of antitumor agents.

Furthermore, as shown in example 10, we have studied on an actionagainst vascular cells, for which an action of the molecules of thepresent invention has never been known except for blood cells, and foundthat the molecules of the present invention have an action to suppressgrowth of the human vascular endothelia cells. Consequently, the presentinvention includes growth suppressive agents for vascular cells andtherapeutic agents for disease (refer to Folkman and Klagsbrun, SCIENCE235, 442-447, 1987), which effect is expected by suppressingvascularization, containing polypeptides having amino acid sequence ofSEQ ID NO: 1-3. The molecules of the present invention can be used fortreatment of these diseases.

In pharmaceutical use, polypeptides of the present invention havingabove form is lyophilized with adding preferable stabilizing agents suchas human serum albumin, and is used in dissolved or suspended conditionwith distilled water for injection when it is in use. For example,preparation for injection or infusion at the concentration of 0.1-1000μg /ml may be provided. A mixture of the compound of the presentinvention 1 mg/ml and human serum albumine 5 mg/ml divided in a vialcould maintain activity of the said compound for long term. Forculturing and activating cells in vitro, lyophilized preparations orliquid preparations of the polypeptide of the present invention areprepared and are added to the medium or immobilized in the vessel forculture. Toxicity of the polypeptide of the present invention wastested. Any polypeptide, 10 mg/kg was administered intraperitoneally inmice, but no death of mice was observed.

In vitro physiological activity of the polypeptide of the presentinvention can be evaluated by administering to disease model mice or itsresembled disease in rats or monkeys, and examining recovery of physicaland physiological functions and abnormal findings. For example, in caseof searching abnormality in relation to hemopoietic cells, bone marrowsuppressive model mice are prepared by administering 5-FU series ofantitumor agents, and bone marrow cell counts, peripheral blood cellcounts and physiological functions are examined in the administeredgroup or the non administered group of mice. Furthermore, in case ofsearching in vitro cultivation and growth of hemopoieticundifferentiated cells including hemopoietic stem cells, the bone marrowcells of mice are cultured in the groups with or without addition of thecompound of the present invention, and the cell cultures are transferredinto the lethal dose irradiated mice. Result of recovery is observedwith the indications of survival rate and variation of blood counts.These results can be extrapolated to the humans, and accordingly usefuleffective data for evaluation of the pharmacological activities of thecompound of the present invention can be obtained.

Applications of the compound of the present invention forpharmaceuticals include diseases with abnormal differentiation of cells,for example leukemia and malignant tumors. These are a cell therapy,which is performed by culturing human derived cells in vitro whilemaintaining their original functions or adding new functions, and atherapy, which is performed by regenerating without damage the functionsoriginally existing in the tissues by administering the compound of thepresent invention under the regeneration after tissue injury. Amount ofadministration may differ in the type of preparation and ranges from 10μg/kg to 10 mg/kg.

Further strong physiological activity can be achieved by expressionforming a multimer of the polypeptide of the present invention. HumanSerrate-2 having multimer structure can be produced by a method ofexpressing chimera protein with human IgG Fc region as described in theexample and expressing the multimer having disulfide bond with hingeregion of the antibody, or a method expressing chimera protein, in whichantibody recognition region is expressed in the C-terminal orN-terminal, and reacting with the polypeptide containing extracellularpart of the thus expressed said human Serrate and the antibody whichrecognize specifically the antibody recognition region in the C-terminalor N-terminal.

Among other methods, a method in which the fused protein bound with onlythe hinge region of the antibody is expressed and the dimer is formed byconstructing with disulfide bond, can be mentioned. A multimer of humanSerrate-2 having higher specific activity than the dimer can beobtained. The said multimer is constructed by fused protein which isprepared for expressing the peptide in the C-terminal, N-terminal orother region. The protein is prepared by forming a disulfide bondwithout affecting any ether activities of the human Serrate-2. Themultimer structure can also be expressed by arranging one or morepeptides containing SEQ ID NOS: 1 or 2, with genetic engineering methodin series or in parallel. Other known methods for providing multimerstructure having dimer or more can be applied. Accordingly, the presentinvention includes any polypeptides containing SEQ ID NO: 1 or 2 in adimer or higher structure prepared by genetic engineering techniques.

As another method, multimerization method using chemical cross-linkercan be mentioned. For example, dimethylsuberimidate dihydrochloride forcross-linking lysine residue, N-(γ-maleimidebutyryloxy) succinimide forcross-linking thiol group of cysteine residue and glutaraldehyde forcross-linking between amino groups can be mentioned. A multimer withdimer or higher structure can be synthesized by applying thesecross-linking reactions. Accordingly, the present invention includes anypolypeptides containing SEQ ID NO: 1 or 2 in the form of dimer or higherstructure prepared by chemical cross-linking agents.

In application of medical care in which cells are proliferated andactivated in vitro and are returned to the body, human Serrate-2 of theform hereinabove can be added directly in the medium, but immobilizationcan also be made. Immobilization method includes applying amino group orcarboxyl group in the human Serrate-2, using suitable spacers or theabovementioned cross-linkers, and the ligand can be covalently bound tothe culture vessels. Accordingly, the present invention includes anypolypeptides containing SEQ ID NO: 1 or 2 in the form existing on asolid surface. The human Serrate-2 molecule binds specifically withreceptor, a Notch receptor molecule. For example, expression of Notchreceptor can be detected by using fused protein with above extracellularregion of the human Serrate-2 and human IgGFc. Notch is known to beinvolved in some types of leukemia (Elissen et al., Cell 66, 649-661,1991). Accordingly, the polypeptides having SEQ ID NO:1 or 2 can be usedfor diagnostic reagents for in vitro or in vivo.

Antibody specifically recognizing the said human Serrate-2 can beprepared as shown in Example 7. Also it can be prepared by variousmethods described in the literature (Antibodies a laboratory manual, E.Harlow et al., Cold Spring Harbor Laboratory), and by recombinantantibody expressed in cells using immunoglobulin gene isolated by amethod of gene cloning. These antibodies can be used for purification ofhuman Serrate-2. Namely, detection and measurement of the humanSerrate-2 of the present invention can be performed by using antibody,which specifically recognizes the humanSerrate-2 shown in Example 7, andcan be applied as diagnostic agent for diseases such as malignant tumoraccompanied with abnormal cell differentiation.

EXAMPLES

The following examples illustrate embodiments of the present invention,but are not to be construed as limiting.

Referential Example 1

Preparation of Human Serrate-1 Gene Probe

A mixed primer corresponding to amino acid sequence conserved inDrosophila Serrate and rat jagged, i. e. sense primer (SEQ ID NO: 8) andantisense primer (SEQ ID NO: 9), were used. The signals used in thesesequence show each equivalent mixture: i.e. S: C and G, Y: T and C, W: Tand A, K: G and T, R: A and G, N: C, G, T and A.

A synthetic oligonucleotide was prepared by using automatic DNAsynthesizer with the principal immobilized method. The automatic DNAsynthesizer used was 391PCR-MATE of Applied Biosystems Inc., U.S.A.Nucleotide, carrier immobilized with 3′-nucleotide, solution andreagents are used according to the instructions by the same corporation.Oligonucleotide was isolated from the carrier after finishing thedesignated coupling reaction and treating the oligonucleotide carrier,from which protective group of 5′-terminal was removed, withconcentrated liquid ammonia at room temperature for one hour. Forremoving protective groups of nucleic acid and phosphoric acid, thereactant solution containing nucleic acid was allowed to stand in theconcentrated ammonium solution in the sealed vial at 55° C. for over 14hours. Each oligonucleotide, from which the carrier and protectivegroups were removed, was purified by using OPC cartridge of the AppliedBiosystems Inc., and detritylated by using 2% trifluoroacetic acid.Primer was dissolved in deionized water to set final concentration 100pmol/μl after purification, and used for PCR. Synthesis ofoligonucleotide was performed by the same manner.

Amplification by PCR was performed as follows.

Human fetal brain originated cDNA mixture solution (QUICK-Clone cDNA,CLONTECH Inc., U.S.A.) 1 μl was used. 10×buffer solution [500 mM KCl,100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , 0.01% gelatin ] 5 μl, dNTPMixture (Takara Shuzo) 4 μl, sense primer DLTS1 (100 pmol/μl ) 5 μl andantisense primer DLTA2 (100 pmol/μl) 5 μl which were specific to theabove Serrate homologue and TaqDNA polymerase (AmpliTaq, Takara Shuzo.,Japan, 5 U/μl) 0.2 μl were added thereto, and finally deionized waterwas added to set up total 50 μl. PCR was performed for 5 cycles of acycle consisting of treatment at 95° C. for 45 seconds, at _(42°) C. for45 seconds and 72° C. for 2 minutes, further 35 cycles of a cycleconsisting of treatment at 95° C. for 45 seconds, at 50° C. for 45seconds, and 72° C. for 2 minutes, and finally allowed to stand at 72°C. for 7 minutes. A part of the PCR products was subjected to 2% agarosegel electrophoresis, stained with ethidium bromide (Nippon Gene Co.,Japan), and observed under ultraviolet light to confirm amplification ofabout 500 bp cDNA. The total amount of the thus obtained PCR product wassubjected to electrophoresis with 2% agarose gel prepared with lowmelting point agarose (GIBCO BRL Inc., U.S.A.), stained with ethidiumbromide, cutting out about 500 bp bands under the UV light, addingdistilled water of the same volume as the gel, heating at 65° C. for 10minutes, and completely dissolving the gel. The dissolved gel wascentrifuged at 15000 rpm for 5 minutes to separate supernatant solutionafter adding an equal volume of TE saturated phenol (Nippon Gene Co.,Japan) and the same separation operation was performed after adding TEsaturated phenol : chloroform (1:1) solution and chloroform. DNA wasrecovered from the final solution by ethanol precipitation.

A vector, pCRII vector (Invitrogen Inc., U.S.A., hereinafter designatedas pCRII) was used. The vector and the above DNA in a molar ratio of 1:3were mixed and DNA was ligated into the vector by using T4 DNA ligase(Invitrogen Inc., U.S.A.). The pCRII, to which DNA was integrated, wassubjected to gene transduction into E. coli one shot competent cells(Invitrogen Inc., U.S.A.) and was spread on the semi-solidmedium plateof L-Broth (Takara Shuzo Co., Japan) containing ampicillin (Sigma Corp.,U.S.A.) 50 μg/ml and allowed to stand at 37° C. for about 12 hours. Thevisible colonies were randomly selected, inoculated in the L-Brothliquid medium 2 ml containing same concentration of ampicillin and shakecultured at 37° C. for about 18 hours. The cultured bacterial cells wererecovered and the plasmid was separated by using Wizard Miniprep(Promega Inc., U.S.A.) according to the attached explanation sheet. Theplasmid was digested by restriction enzyme EcoRI. Integration of thesaid PCR product was confirmed by incision of about 400 bp DNA. The basesequence of the incorporated DNA in the confirmed clone was determinedby fluorescent DNA sequencer (Model 373S, Applied System Inc., U.S.A.).The gene cloning gene fragment was compared with amino acid sequence ofNotch ligand molecule, i.e. Drosophila Serrate and rat Jagged, andsignificant analogous sequence was found, then the sequence wasconfirmed as cDNA fragment coding human Serrate-1.

Referential Example 2

Cloning of Full Length Human Serrate-1 Gene

A screening of clones having full length cDNA was performed byhybridization from human placenta origin cDNA library (inserted cDNA inλgt-11, CLONTEC Inc., U.S.A.) in plaques corresponding to 1×106 plaques.Generated plaques were transcribed to nylon filter (Hybond N+: AmershamInc., U.S.A.). The transcribed nylon filter was subjected to alkalinetreatment [allow to stand for 7 minutes on a filter paper permeated witha mixture of 1.5 M NaCl and 0.5 M NAOH], followed by twice neutralizingtreatments [allow to stand for 3 minutes on a filter paper permeatedwith a mixture of 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2) and 1 mM EDTA].Subsequently, the nylon filter was shaken for 5 minutes in 2-foldconcentrated SSPE solution [0.36 M NaCl, 0.02 M sodium phosphate (pH7.7) and 2 mM EDTA], washed, and air-dried. Then the nylon filter wasallowed to stand for 20 minutes on a filter paper, which was permeatedwith 0.4 M NAOH, and was shaken for 5 minutes with 5-fold concentratedSSPE solution and was washed, then again air-dried. Screening wasconducted in the human Serrate-1 probe labeled with radioisotope 32 pusing the filter.

The DNA probe previously prepared was labeled with 32 p as follows. ADNA fragment was cut out by EcoRI from PCR II, to which purified PCRproduct by human Serrate primers (about 500 bp) was inserted and DNAfragments were isolated from low melting point agarose gel. The thusobtained DNA fragment was labeled by DNA labeling kit (Megaprime DNAlabeling system: Amersham, U.S.A.). The primer solution 5 μl anddeionized water were added to DNA 25 ng to set up total volume of 33 μl,which was treated for 5 minutes in boiling water bath. Reaction buffersolution 10 μl containing NTP, α-32 P-dCTP 5 μl and T4 DNApolynucleotide kinase solution 2 μl were added thereto, and treated at37° C. for 10 minutes in water bath. Subsequently, the mixture waspurified by Sephadex column (Quick Spin Column Sephadex G-50: BoehringerMannheim Inc., Germany), then treated for 5 minutes in boiling waterbath and ice-cooled for 2 minutes for use.

Hybridization was performed as follows. The prepared filter hereinabovewas immersed into prehybridization solution consisting of SSPE solution,in which final concentration of each component is set at 5-foldconcentration, 5-fold concentration of Denhardt's solution (WakoPurechemicals), 0.5% SDS (sodium dodecyl sulfate) and salmon sperm(Sigma Co.) 10 μg/ml denatured by boiling (Sigma Co.) 10 μg/ml denaturedby boiling water, shaken at 65° C. for 2 hours, then the filter wasimmersed into the hybridization solution, which was the same compositionas the above prehybridization solution, containing the probe labeledwith 32 P by the above mentioned method, and shaken at 55° C. for 16hours to perform hybridization.

The filter was washed by immersing into SSPE solution containing 0.1%SDS, shaken at 55cC twice, and further immersing into 10-fold dilutionof SSPE solution containing 0.1% SDS four times at 55° C. The washedfilter was treated with autoradiography using a sensitized screen.Clones of strongly exposed part were collected and plaques obtained wereagain spread and screened by the same method hereinbefore to separate acomplete single clone.

The thus isolated phage clones were 22 clones. Phages of all of theseclones was prepared to about 1×10 9 pfu, whereafter the phage DNA waspurified, digested by restriction enzyme EcoRI and inserted intopBluescript (Stratagene Inc., U.S.A.), which was digested by EcoRI inthe same way. DNA sequences of both ends of these clones were analyzedby DNA sequencer Two clones of S16 and S20 were the clone containing DNAsequence from No. 1 to 1873 in the sequence listing, SEQ ID NO: 6.Clones S5 and S16 were the clone containing DNA sequence from No. 990 to4005 in the sequence listing, SEQ ID NO: 6. The deletion mutant of theseclones were prepared by using kilosequence deletion kit (Takara ShuzoCo.) according to a description of the attached paper. The full-lengthcDNA base sequence encoding a polypeptide of the present invention wasdetermined using the DNA sequencer (Applied Biosystem Inc.) from bothdirection of 5′-direction and 3′-direction.

As a result, about 100 bp in an area coding C-terminal amino acidsequence were found to be not cloned, accordingly cloning of full-lengthgene was performed by using GIBCO-BRL, 3′RACE system kit according tothe attached manual. Namely, cDNA cloning was performed by human originpoly A+RNA (CLONTECH Corp.) to 3′-direction and gene sequence wasdetermined.

The thus cloned three gene fragments in a plasmid containing in afull-length DNA sequence of SEQ ID NO: 6 are inserted by applyingrestriction enzyme Bgl 2 site at DNA sequence No. 1293 in sequence IDNO: 6 and AccI site at No. 3943, between EcoRI and XbaI of multicloningsite in pUC18 to prepare pUCSR-1. The sequence of this gene togetherwith aminoacid sequence is shown in SEQ ID NO: 6.

Example 1

Preparation of Probe by PCR

Gene probes used for screening, i.e. gene described in SEQ ID NO: 10, 11and 12, were obtained as follows. These sequences correspond to Genbankregistered number, TO8853, R50026 and R45751. Hereinafter, a probehaving gene sequence SEQ ID NO: 10 is designated as ¥1, a probe havinggene sequence SEQ ID NO: 11 is designated as ¥2, and a probe having genesequence SEQ ID NO: 12 is designated as ¥4.

Namely, a gene SEQ ID NO: 10 was isolated by PCR using primers ofoligonucleotide having SEQ ID NOS: 13 and 14; a gene SEQ ID NO: 11 wasisolated by PCR using primers of oligonucleotide having SEQ ID NO: 15and 16; and a gene SEQ ID NO: 12 was isolated by PCR using primers ofoligonucleotide having SEQ ID NO: 17 and 18.

Amplification by PCR was performed as follows. Human fetal brainoriginated cDNA mixed solution (QUICK-Clone cDNA, CLONTECH Inc., U.S.A.)1 μl was used. 10×buffer solution [500 mM KCl, 100 mM Tris-HCl (pH 8.3),15 mM MgCl 2, 0.01% gelatin] 5 μl, dNTP mixture (Takara Shuzo Co.,Japan) 4 μl, the primers hereinbefore (20 pmol/μl) each 1 μl, and TaqDNApolymerase (AmpliTaq, Takara Shuzo Co., 5 U/μl) 0.2 μl were addedthereto, and finally deionized water was added to set up total 50 μl.PCR was performed for 40 cycles of a cycle consisting of treatment at95° C. for 1 minute, at 550° C. for 5 minutes and 72° C. for 3 minutes,and finally allowed to stand at 72° C. for 7 minutes. A part of the PCRproducts was subjected to 2% agarose gel electrophoresis, stained withethidium bromide (Nippon Gene Co., Japan), and observed underultraviolet light to confirm amplification of objective size gene.

The total amount of the thus obtained PCR product was subjected toelectrophoresis with 2% agarose gel prepared with low melting pointagarose (GIBCO BRL Inc., U.S.A.), stained with ethidium bromide, cuttingout each band under the UV light, adding distilled water of the equalvolume of the gel, heating at 65° C. for 10 minutes, and completelydissolving the gel. The dissolved gel was centrifuged at 15000 rpm for 5minutes to separate supernatant solution after adding equal volume of TEsaturated phenol (Nippon Gene Co., Japan) and the same separationoperation was performed after adding TE saturated phenol:chloroform(1:1) solution and chloroform. DNA was recovered from the final solutionby ethanol precipitation.

A vector, pCRII vector (Invitrogen Inc., U.S.A., hereinafter designatedas PCRII) was used. The vector and the above DNA in a molar ratio 1:3were mixed and DNA was ligated into the vector by using T4 DNAligase(Invitrogen Inc., U.S.A.). The PCRII, to which DNA was integrated, wassubjected to gene transduction in E. coli one shot competent cells(Invitrogen Inc., U.S.A.) and was spread on the semi-solid medium plateof L-Broth (Takara Shuzo Co., Japan) containing ampicillin (Sigma Corp.,U.S.A.) 50 μg/ml and allowed to stand at 37° C. for about 12 hours. Thevisible colonies were randomly selected, inoculated in the L-Brothliquid medium 2 ml containing the same concentration of ampicillin, andshake cultured at 37° C. for about 18 hours. The cultured bacterialcells were recovered and the plasmid was separated by using WizardMiniprep (Promega Inc., U.S.A.) according to the attached explanationsheet. The plasmid was digested by restriction enzyme EcoRI. Integrationof the said PCR product was confirmed by incision of objective size DNA.The base sequence of the incorporated DNA in the confirmed clone wasdetermined by fluorescent DNA sequencer (Model 373S3, Applied SystemInc., U.S.A.). The sequence was compared with DNA sequence registered inGenbank hereinbefore. Isolation of gene having DNA sequences SEQ ID NO:10, 11 and 12 was confirmed.

Example 2

Cloning of Full Length Human Serrate-2 Gene

A screening of clones having full length cDNA was performed byhybridization from human fetal brain origin cDNA library (inserted cDNAin λgt-11, CLONTECH Inc.) in plaques corresponding 1×10 6 plaques.Appeared plaques were fixed with alkali by the same method as describedin Referential example 2. Using these filter and three types of probes,which were isolated in Example 1 and labeled with 3 2 p by the methoddescribed in Referential example 2, screening was performed on eachindividually to obtain clones.

Isolated phage clones were: 2 clones from a case using ¥1 as a probe; 6clones from a case using ¥2 as a probe; and 4 clones from a case using¥4 as a probe. Clones isolated by ¥4 were all included in clonesisolated from ¥2. All of the phages of these clones were prepared toabout 1×10 9 pfu, and phage DNA was purified by using Wizard Lambdapreps (Promega Inc.) according to the attached explanation sheet, anddigested by EcoRI, then incorporated into pBlusecript (Stratagene Inc.)or pUC18 (Pharmacia Inc.) digested by EcoRI.

The full DNA sequences of these clones were determined by DNA sequenceras same as Referential example 2, and a part of the identical sequencewas compared. Result indicates that: #5 clone was a clone containing DNAsequence from No. 484 to 2025 in sequence ID NO: 4; #21 clone was aclone containing DNA sequence from No. 1882 to 3537 in SEQ ID NO: 4; and#86 clone was a clone containing DNA sequence from No. 2455 to 3955 inSEQ ID NO: 4. Remaining clones were those having only short insertconsisting of a part identical with sequences of these clones. Thisresult indicated that, as a result of comparison with amino acidsequences of human Serrate-1, the area encoding the N-terminal aminoacid sequence was not cloned. Consequently, a probe having DNA sequenceof SEQ ID NO: 19 was prepared and a screening of the second time wasperformed for cloning the cDNA in 5′-region. A probe was prepared assame as described in Example 1, namely PCR primers having DNA sequenceof SEQ ID NO: 20 and 21 were subjected to PCR using #5 clone as atemplate. Library used was prepared as same as the first screening andconditions were performed by the same method as before.

The thus isolated clones in the second screening were six clones. Phagesof all these clones were prepared about 1×10 9 pfu, purified by usingWizard Lambda Preps (Promega Inc.) according to the attached explanationsheet, digested by EcoRI and inserted into pUC 18 which was alsodigested with EcoRI. The full DNA sequences of these clones weredetermined by DNA sequence as same as in Referential example 2. As aresult of comparison with a part of identical sequence, S43-1 clone wasconsidered to contain the most 5′-direction. This clone was a clonecontaining DNA sequence from Nos. 38 to 1538 in SEQ ID NO: 4. Theremaining clones have only short inserts consisting of a part identicalwith sequence determined already in the other clone or isolated in thefirst time.

Although a sequence of ATG, which codes translation initiation codonmethionine, could not be found in the second screening clones, furthercloning of cDNA sequence for 5′-direction was performed by 5′RACEmethod. 5′RACE was performed by using 5′RACE system kit (GIBCO-BRL Inc.)according to the attached manual. A cloning of cDNA with 5′-direction inthe gene using human heart origin poly A+RNA (CLONTECH Inc.) wasperformed, and a gene sequence from DNA sequence No. 1 to No. 37 in SEQID NO: 4 was determined.

As a result, DNA sequence in SEQ ID NO: 4, i. e. cDNA sequence encodingfull length of human Serrate-2, was determined.

In order to prepare cDNA encoding the full-length gene, and to obtaincDNA of the 5′-terminal, which could ligate with other clones, thefollowing PCR was performed for cloning. Namely, using theoligonucleotide having DNA sequence in SEQ ID NO: 22 and theoligonucleotide having DNA sequence in SEQ ID NO: 23, PCR was performedusing S43-1 clone as a template according to a method described inExample 1. Similarly, it was subcloned to pCRII and gene sequence wasdetermined to prepare the clone having DNA sequence from Nos. 1 to No.503 in SEQ ID NO: 4. This clone is designated as S2-5.

Among the above clones, i.e. gene S2-5, S43-1, #5, #21 and #86, S2-5 andS43-1 were applied in the restriction enzyme Spl I site at DNA sequenceNo. 217 in SEQ ID NO: 4; S43-1 and #5 were the same as in Kpn I site atNo. 1453; #5 and #21 were the same as in Sac I site at No. 2016; and #21and #86 were the same as in BamHI site at No. 2991, and finally DNAhaving DNA sequence SEQ ID NO: 4 was inserted between EcoRI site andHind III site of multi cloning site of pUC 18 to prepare pUCSR-2.

Example 3

Expression of Human Serrate-2 in Organs

In order to examine expression of mRNA of human Serrate-2, usingfilters, which was previously transcribed with mRNA, i.e. Human MultipleTissue Northern Blot, Human Multiple Tissue Northern Blot II, HumanMultiple Tissue Northern Blot III and Human Fetal Multiple TissueNorthern-Blot II (CLONTECH Inc.), 32 p labeling was performed by theprevious method using DNA labeling kit (Mega Prime DNA lebeling system:Amersham Inc.) hereinbefore mentioned, with a probe DNA having SEQ IDNO: 19 described in Example 2, and expression was examined withperforming hybridization according to description of instruction for useattached to the above filters.

As a result, length of expressed mRNA was about 5 kb. Strong expressionsin human adult tissues were observed in heart, skeletal muscle, thyroidgland, spinal cord and trachea; clear expression was observed inpancreas, prostate, testis, small intestine and adrenal gland, very weakexpression was observed in brain, placenta, kidney, thymus, ovary,stomach and lymph node, and no expression was observed in lung, liver,spleen, colon, peripheral lympocytes and bone marrow. In the human fetaltissues, strong expression was observed in the fetal lung, clearexpression: fetal brain and fetal kidney and no expression in fetalliver.

Example 4

Preparation of Expression Vector of Human Serrate-2

Using the gene consisting of DNA sequence described in the sequencelisting, SEQ ID NO: 4, expression vectors of human Serrate-2 and itschimera protein mentioned in the following 1) to 5) were prepared.

1) Expression Vector of Secretory Extracellular Human Serrate-2

The cDNA coding polypeptide of amino acid sequence from No. 1 to 1055 inthe sequence listing, SEQ ID NO: 2 was ligated with expression vectorpMKITNeo (Maruyama et al., Preliminary Paper, Japan Molecular BiologySoc.1991, obtainable from Prof. Maruyama, Tokyo Medical and DentalUniv.), which has a SR α promoter and Neomycin resistant gene, toprepare expression vector.

Namely, vector pUCSR-2 containing DNA sequence in SEQ ID NO: 4 was usedas template and oligonucleotide having sequence in SEQ ID NO: 26 andoligonucleotide having sequence in SEQ ID NO: 27 were used as primer,and PCR was performed according to a method described hereinbefore. ThePCR product was ligated into cloning vector PCRII, whereupon the genesequence of the PCR product was determined to prepare DNA having genesequence from No. 2986 to 3254 of DNA sequence in SEQ ID NO: 4, in whichtermination codon and restriction enzyme Sal I site were attached in the3′ end.

About a 3 kbp gene fragment was obtained by digesting the pUCSR-2 withrestriction enzyme EcoRI and BamHI, about a 250 bp gene fragment wasobtained by digesting the pCRII vector with restriction BamHI and Sal I,the vector of which contained the above PCR product as an insert, andabout a 4.3 kb gene fragment was obtained by digesting the pMKITNeo withrestriction enzymes EcoRI and XhoI, and these 3 gene fragments weresimultaneously ligated to obtain the expression vector containing genefragment of DNA sequence from No. 1 to 3254 in SEQ ID NO: 4. Secretoryextracellular human Serrate-2 protein (hereinafter designated thisprotein as EXS2) expression vector pMEXS2 was obtained.

2) Expression Vector of FLAG Chimera Protein of Secretory ExtracellularHuman Serrate-2

The cDNA coding chimera protein, to which cDNA coding FLAG sequence (SEQID NO: 24) was added to the C-terminal of polypeptide from No. 1 to 1055of amino acid sequence in the sequence listing, SEQ ID NO: 2 was ligatedto the expression vector pMKINeo to prepare the expression vector.

Namely, vector pUCSR-2 containing DNA sequence in SEQ ID NO: 4 was usedas template and oligonucleotide having sequence in SEQ ID NO: 26 andoligonucleotide having sequence in SEQ ID NO: 28 were used as primer,and PCR was performed according to a method described hereinbefore. ThePCR product was ligated to cloning vector pCRII, and the gene sequenceof the PCR product was determined to prepare DNA having gene sequencefrom No. 2986 to 3254 of DNA sequence in SEQ ID NO: 4, in which DNAsequence coding FLAG sequence in the 3′ end (DNA sequence in SEQ ID NO:24), termination codon and restriction enzyme Sal I site were attachedin the 3′ end.

About a 3 kbp gene fragment was obtained by digesting the pUCSR-2 withrestriction enzyme EcoRI and BamHI, about a 300 bp gene fragment wasobtained by digesting the pCRII vector with restriction enzymes BamHIand Sal I, the vector of which contained the above PCR product as aninsert, and about a 4.3 kb gene fragment was obtained by digesting thepMKITneo with restriction enzymes EcoRI and XhoI, and these 3 genefragments were simultaneously ligated (though recognition sequence ofrestriction enzymesXho I and Sal I is different, they can be ligated dueto complementary terminal gene sequence) to obtain the expression vectorcontaining gene fragment of DNA sequence from No. 1 to 3254 in SEQ IDNO: 4 and gene fragment encoding FLAG sequence. Secretory extracellularhuman Serrate-2 FLAG chimera protein (hereinafter designated thisprotein as EXS2FLAG) expression vector pMEXS2FLAG was obtained.

3) Expression Vector of IgG1Fc Chimera Protein of SecretoruyExtracellular Human Serrate-2

A cDNA coding chimera protein, to which cDNA coding amino acid sequenceof Fc region below the hinge part of human IgG1 was added to theC-terminal of polypeptide having amino acid sequence from No. 1 to 1055of in the sequence listing, SEQ ID NO: 2, was ligated to the expressionvector pMKINeo to prepare the expression vector. Peparation of fusedprotein with immunoglobulin Fc protein was performed according to themethod of Zettlmeissl et al. (Zettlmeissl et al., DNA cell Biol., 9,347-354, 1990). A gene using genome DNA with intron was applied and thesaid gene was prepared by using PCR.

Human genomic DNA was used as a template. Oligonucleotide of thesequence in the sequence listing, SEQ ID NO: 31 with restriction enzymeBamHI site, and oligonucleotide of the sequence in the sequence listing,SEQ ID NO: 32 with restriction enzyme XbaI site were used as primer. PCRof gene sequence encoding human IgG1F was performed using the primersand human genomic DNA as template. About 1.4 kbp band was purified,treated by restriction enzyme BamHI and XbaI (Takara Shuzo Co., Japan),and genes were ligated to pBluescript, which was similarly treated byrestriction enzyme, by using T4 DNA ligase to prepare subdloning. Later,the plasmid DNA was purified and sequenced to confirm gene sequence,then the said gene sequence was confirmed as genome DNA in the hingeregion of heavy chain of the human IgG1. (The sequence is referred toKabat et al., Sequence of Immunological Interest, NIH Publication No.91-3242, 1991). Hereinafter this plasmid is designated as pBShIgFc.

A vector pUCSR-2 having DNA sequence in SEQ ID NO: 4 was used astemplate and oligonucleotide having sequence in SEQ ID NO: 26 andoligonucleotide having sequence in SEQ ID NO: 29 were used as primer,and PCR was performed according to a method described hereinbefore. ThePCR product was ligated to cloning vector pCRII, and the gene sequenceof the PCR product was determined to prepare DNA having gene sequencefrom No. 2986 to 3254 of DNA sequence in SEQ ID NO: 4 in whichrestriction Bgl 2 site was attached in the 3′ end.

About a 250 bp gene fragment wasobtained by digesting the PCRII vector,which contained the above PCR product as an insert, with restrictionenzymes EcoRI and BamHI containing a gene encoding the human IgG1FC asan insert were ligated. In this case, BamHI and Bgl 2 sites can beligated due to complementary of the digested terminal gene sequence.Further this part cannot be digested by these restriction enzymes.

About a 1.5 kbp gene fragment was obtained by digesting this vector withrestriction enzyme BamHI and Not 1, about a 3 kbp gene fragment wasobtained by digesting PUCSR-2 with restriction enzymes EcoRI and BamHI,and about a 4.3 kb gene fragment was obtained by digesting pMKITneo withrestriction enzyme EcoRI and Not 1, and these 3 gene fragments weresimultaneously ligated (though restriction enzymes Xho I and Sal I havedifferent recognition sequence, they can be ligated due to complementaryterminal gene sequence). An expression vector containing gene fragmentfrom DNA sequence No. 1 to 3254 in SEQ ID NO: 4 and a gene fragmentcoding human IgG1Fc, expression vector pMEXS2Fc of Ig chimera protein ofsecretory extracellular humanSerrate-2 (hereinafter this protein isdesignated as EXS2Fc), was obtained.

4) Expression Vector of Full Length Human Serrate-2 Protein

The cDNA coding polypeptide from No. 1 to 1212 of amino acid sequenceinthe sequence listing, SEQ ID NO: 3, was ligated to the expression vectorpMKITNeo to prepare the expression vector.

Namely, about 4 kbp gene fragment, which was cut out by digestingpUCSR-2 with restriction enzymes EcoRI and Hind III, was ligated intopBluescript, which was digested with the same restriction enzymes.Subsequently, about 4 kbp gene fragment cut from this vector bydigesting with EcoRI and XhoI was ligated with about 4.3 kb genefragment obtained by digesting the expression vector pMKITneo withrestriction enzymes EcoRIand Not 1 to prepare expression vectorcontaining gene fragment of DNA sequence from No. 1 to 3955 in SEQ IDNO: 4. Full length human Serrate-2 protein (hereinafter this protein isdesignated as FS2) expression vector pMFS2 was obtained.

5) Expression Vector of FLAG Chimera Protein of Full Length HumanSerrate-2

The cDNA coding chimera protein, to which cDNA coding FLAG sequence (SEQID NO: 24) was added to the C-terminal of polypeptide from No. 1 to 1212of amino acid sequence in the sequence listing, SEQ ID NO: 3, wasligated to the expression vector pMKITNeo to prepare the expressionvector.

Namely, vector pUCSR-2 having DNA sequence in SEQ ID NO: 4 was used astemplate and oligonucleotide having sequence in SEQ ID NO: 26 andoligonucleotide having sequence in SEQ ID NO: 30 were used as primer,and PCR was performed according to a method described hereinbefore. ThePCR product was ligated to cloning vector PCRII, and the gene sequenceof the PCR product was determined to prepare DNA having gene sequencefrom No. 2986 to 3725 of DNA sequence in SEQ ID NO: 4, in which DNAsequence coding FLAG sequence in the 3′ end (DNA sequence in SEQ ID NO:24), termination codon and restriction enzyme Sal I′site were attachedin the 3′ end.

About a 3 kbp gene fragment was obtained by digesting the pUCSR-2 withrestriction enzymes EcoRI and BamHI, about a 700 bp gene fragment wasobtained by digesting the PCRII vector by restriction enzymes BamHI andSalI, the vector of which contained the above PCR product vector ofwhich contained the above PCR product as an insert, and about a 4.3 kbgene fragment was obtained by digesting the pMKITneo with restrictionenzymes EcoRI and XhoI, and these 3 gene fragments were simultaneouslyligated (though recognition sequence of restriction enzymes XhoI andSalI is different, they can be ligated due to complementary terminalgene sequence) to obtain the expression vector containing gene fragmentof DNA sequence from No. 1 to 3725 in SEQ ID NO: 4 and gene fragmentcoding FLAG sequence. Full length human Serrate-2 FLAG chimera protein(hereinafter designated this protein as FS2FLAG) expression vectorpMFS2FLAG was obtained.

Example 5

Expression and Gene Transfer of the Human Serrate-2 Expression Vectorsinto Cells

The expression vectors prepared in Example 4 were gene transduced intoCOS-7 cells (obtained from RIKEN Cell Bank, Physical and ChemicalResearch Institute, Japan, RCB0539).

Cell culture before transduction was performed by culturing in D-MEM(Dulbecco modified Eagle's medium, GIBCO-BRL Inc., U.S.A.) 10% FCS. Onthe day before gene transduction, medium of cells was changed to setcell counts 5×10 7 cells/ml and cultured overnight. On the day of genetransduction, cells were sedimented by centrifugation, centrifugallywashed twice with PBS (−) and prepared to 1×10 7 cells/ml in PBS (−), 1mM MgCl 2 and gene transfer was performed by electroporation using genetransduction device Gene-pulsar (Bio-Rad Inc., U.S.A.). The above cellsuspension 500 μl was collected in the cell for electroporation (0.4mm), expression vector 20 μg was added, and allowed to stand in ice for5 minutes. Electroporation was performed under the condition 3 μF, 450Vtwice, and during the two electroporations the cell mixture was allowedto stand at room temperature for 1 minute. After 5 minutes in ice, cellswere spread in the culture medium, diameter 10 cm previously added with10 ml of the medium described hereinbefore, and cultured at 37° C. in 5%carbon dioxide incubator.

The next day, the culture supernatant solution was removed, the cellsadhered to the dish were washed twice with PBS (−) 10 ml and serum-freeD-MEM 10 ml was added and cultured for 4 days. In case of genetransduction into expression vectors pMEXS2, pMEXS2FLAG and pMEXS2Fc,culture supernatant solution was recovered and was replaced the bufferto PBS (−) by Centricon 30 (Amicon Inc., U.S.A.) and simultaneously thesolution was concentrated to 10-fold to obtain cell culture supernatantsolution.

In case of gene transduction of pMSF2 and pMFS2FLAG, after 4 daysculture, cells were washed with PBS (−) 10 ml. Cells were scraped usingcell scraper (Corster Corp.), PBS (−) 10 ml was added again, centrifugedat 1500 rpm for 5 minutes and washed. Cell precipitates were suspendedin the cell lysis buffer [50 mM Hepes (pH 7.5), 1% Triton X-100, 10%glycerol, 4 mM EDTA, 50 μg/ml Aprotinin, 100 μM Leupeptin, 25 μMPepstatin A and 1 mM PMSF] 500 μl, allowed to stand in ice for 20minutes and centrifuged at 15000 rpm for 20 minutes to collectsupernatant solution to obtain cell lysates.

Using these samples, expression of FLAG chimera and immunoglobulinchimera proteins were detected by Western blotting. Namely, concentratedcultured supernatants or cell lysates were subjected to SDS-PAGE usingan electrophoresis tank and polyacrylamide gel for SDS-PAGE (gradientgel 5-15%) (ACI Japan Inc.) according to manufacturer's construction.Samples were prepared by treatment in boiling water for 5 minutes with2-mercapto-ethanol (2-ME) for reduction, and non-reduced conditionwithout taking the above treatment. As a marker, Rainbow Marker (highmolecular weight, Amersham Inc.) was used. Sample buffer solution andelectrophoresis buffer were prepared with reference to the attachedleaflet. When the SDS-PAGE was finished, acrylamide gel was transcribedto PVDF membrane filter (BioRad Inc., U.S.A.) using the Mini Trans BlotCell (BioRad Inc.).

The thus prepared filter was shaken overnight at 4° C. in Blockace(Dainippon Pharm. Co., Japan), TBS-T [20 mM Tris, 137 mM NaCl (pH 7.6)and 0.1% Tween 20] to effect blocking. According to the explanation ofthe attached leaflet of ELC Western blotting detection system (AmershamInc., U.S.A.); in case that protein was FLAG chimera, anti-FLAG M2 mousemonoclonal antibody (Eastman Kodak, U.S.A.) was used as primaryantibody, and peroxidase labeled anti-mouse Ig sheep antibodies(Amersham Inc., U.S.A.) as a secondary antibody, were reacted. In caseof human IgGlFc chimera, peroxidase labeled anti-human Ig sheepantibodies (Amersham Inc., U.S.A.) were reacted. Reaction time forantibodies was 1 hour at room temperature, and at an interval of eachreaction, washing was performed by shaking in TBS-T at room temperaturefor 10 minutes for three times. After the final washing, the filter wasimmersed in the reaction solution of ELC-Western blotting detectionsystem (Amersham Inc.,U.S.A.) for 1 minute, and wrapped inpolyvinylidene chloride wrap for exposure to X-ray film.

As a result, the bands showing protein obtained by transduction ofexpression vector pMEXS2FLAG were detected from COS supernatant about135 kD by anti-FLAGM2 antibody; and production of objective proteinEXS2FLAG was confirmed, and transduced cells by expression vectorpMEXS2FLAG. Molecular weight changes depending on reduction treatment atthe SDS-PAGE were not observed. About 20 kD of sugar chains was added tothe molecules as a result of comparing with a molecular weight estimatedby amino acid sequence.

Furthermore, a band having molecular weight about 165 kD was detectedfrom supernatant solution of COS cells, to which the expression vectorpMEXS2Fc was gene transduced, on SDS-PAGE by anti-human Ig sheepantibody under reducing conditions. A band having molecular weight about330 kD was detected under non-reducing conditions. These resultsindicated that objective protein EXS2Fc was produced, and consequentlytransformed cells by the expression vector pMEXS2Fc could be obtained.As the molecular weight of EXS2Fc under reducing conditions is abouthalf of that under non-reducing conditions, the EXS2Fc is estimated tohave a construction of dimer through disulfide bond. Furthermore, themolecular weight of the band is about 40 kD larger than that calculatedfrom amino acid sequence. This indicates addition of sugar chain to themolecule.

Furthermore, a band having molecular weight about 150 kD was detectedfrom extract of COS cells, to which the expression vector pMFS2FLAG wasgene transduced, on SDS-PAGE by anti-FLAG M2 antibody under reducingconditions. The results indicated that the objective protein FS2FLAG wasproduced, and consequently cells transformed by expression vectorpMFS2FLAG, were obtained. As the molecular weight of FS2FLAG of the bandis about 20 kD larger than that calculated by amino acid sequence, sugarchains may be added to the extracellular region.

As for a protein other than chimera protein, detection was conducted byusing anti-human Serrate-2 mouse monoclonal antibody and anti-humanSerrate-2 rabbit polyclonal antibody, which were described in Example 7,as primary antibodies in the Western blotting. Also as secondaryantibody, anti-mouse Ig sheep antibody (Amersham Inc.) or peroxidaselabeled rabbit Ig sheep antibody (Amersham Inc.) were used.

As a result, a band having molecular weight about 135 kD was detected inthe supernatant of COS cells, to which the expression vector pMEXS2wasgene transduced. This indicated that objective protein EXS2 wasproduced, and cells transformed by an expression vector pMEXS2 could beobtained. No changes of molecular weight were observed caused byreduction treatment on SDS-PAGE. A band having molecular weight about150 kD was detected under reducing conditions from COS cell extract, towhich the expression vector pMFS2 was gene transduced. These resultsindicated that objective protein FS2 was produced, and consequentlycells transformed by the expression vector pMFS2 could be obtained.Furthermore, in every case, molecular weight of the band is about 20 kDlarger than that calculated from amino acid sequence. This indicatesaddition of sugar chains to the molecule of the extracellualr region.

In the control experiments, cell lysate and cultured supernatantsolution of COS-7 cells, to which pMKITNeo vector was transformed, weretested. No bands reacted with anti-FLAG antibody, anti-human Ig antibodyor anti-human Serrate-2 antibody could be detected.

Example 6

Purification of Secretory Extracellular Human Serrate-2 Chimera Proteinsof Gene Transduction Cells

Cultured supernatant of COS-7 cells transformed by the expression vectorpMEXS2FLAG or pMEXS2Fc by a method in Example 5, were prepared in largescale, and chimera protein, i.e. EXS2FLAG or EXS2Fc, was purified byaffinity column chromatography.

In case of EXS2FLAG, 2 liters of the cultured supernatant obtained bythe method in Example 5 was passed through a column packed withAnti-FLAG M2 Affinity Gel (Eastman Kodak, U.S.A.). The chimera proteinwas absorbed in a column by a reaction of affinity of anti-FLAG antibodyof the gel and FLAG sequence of the chimera protein. An inner diameter10 mm, disposable column (BioRad Inc., U.S.A.) was used with packing theabove gel 5 ml. A circulation system consisting of mediumbottle→column→peristaltic pump→medium bottle was set up. The circulationwas run by a flow 1 ml/min. for 72 hours. Thereafter the column waswashed with PBS (−) 35 ml and eluted by 0.5 M Tris-glycine (pH 3.0) 50ml. The eluate of 25 fractions, each 2 ml, was collected into the tube(Farcon Inc.2063), and each fraction was neutralized by 200 μl of 0.5 MTris-HCl (pH 9.5) previously added in each tube.

The eluate fraction, each 10 μl of the EXS2FLAG which was purified bythe above method was subjected to reduction treatment described inExample 5. SDS-PAGE electrophoresis by 5-10% gradient polyacrylamide gelwas performed. After finishing the electrophoresis, silver staining wasconducted by using Wako silver stain kit II according to the explanationof the attached leaflet. Fractions from No. 4 to 8 showed detectablebands in EXS2FLAG. The size is identical with the result of Westernblotting of anti-FLAG antibody obtained in Example 5. Therefore,purified EXS2FLAG was obtained.

In the EXS2Fc, two liters of the cultured supernatant solution wasabsorbed in Protein A Sepharose column (Pharmacia Inc., Sweden)according to the same method as above to collect the eluate fractions.Using a part of eluate as same as in EXS2FLAG, a determination of theeluate fraction, identification of the size and detection of the puritywere performed by SDS-PAGE electrophoresis and silver staining inreducing conditions. Therefore, the eluate fractions from No. 4 to 15were detected as bands. The molecular weight thereof is identical withthe result of Example 5. Therefore, purified EXS2Fc was obtained.

Example 7

Preparation of Antibodies Recognizing Human Serrate-2

EXS2FLAG, purified by the method in Example 6, was used as immunogen,and rabbits were immunized. After assaying antibody titer, whole bloodwas collected and serum was obtained. Anti-human Serrate-2 rabbitpolyclonal antibody were purified by using Econopack serum IgGpurification kit (BioRad Inc., U.S.A.) with reference to the attachedexplanation leaflet.

EXS2FLAG purified by a method described in Example 6 was used asimmunogens, and mouse monoclonal antibodies were prepared according tothe explanation of the textbook. The purified HSFLAG was administered inBalb/c mice (Nippon SLC Co., Japan), 10 μg/mouse, immunizedintracutaneously and subcutaneously. After second immunization,increased serum titer was confirmed by collecting bloodophthalmologically, and the third immunization was performed.Subsequently, the spleen of mice was collected and fused with mousemyeloma cells P3X63Ag8 (ATCC TIB9) using polyethylene-glycol. Hybridomawas selected by HAT medium (Immunological and BiologicalResearchInstitute, Japan), and the hybridoma strains, which produced antibodyspecifically recognizing extracellular region of human Serrate in themedium, were isolated by enzyme immunoassay. The hybridoma strainsproducing mouse monoclonal antibody, which specifically recognized humanSerrate-2, were established.

Anti-human Serrate-2 monoclonal antibody was purified and prepared byusing Mab Traq GII (Pharmacia Inc., Sweden) and according to theexplanation of the leaflet, from the supernatant of the thus establishedhybridoma.

Affinity column chromatography was performed by using the monoclonalantibody. Preparation of the affinity column was performed according tothe explanation attached to the CNBr activated Sephadex 4B (PharmaciaInc., Sweden). Coupling efficiency was 99.6%. A column, 2 cm×1 cm,containing gel 2 ml, was prepared.

A supernatant of the cultured cells, which contained EXS2, was passedthrough the column. The supernatant solution was passed at 20 ml/hr,subsequently PBS (−) 15 ml was passed at the same flow rate and washedthe column. Finally, the products were eluted by a mixture of 0.1 Msodium acetate and 0.5 M NaCl (pH 4.0). The eluate, each 1 ml fractionwas collected, and was neutralized by adding 1 M Tris-HCl (pH 9.1) 200μl for each fraction.

SDS-PAGE of purified protein was conducted under reducing conditionsaccording to the method described in Example 5, followed by silverstaining and Western blotting to estimate molecular weight. A band ofabout 140 kD was detected. Consequently, Western blotting can be made byusing the said monoclonal antibodies and human Serrate-2 can be purifiedby the affinity columns.

Example 8

Effects of Human Serrate-2 Protein on Colony Formation of BloodUndifferentiated Cells

In order to observe physiological action of human Serrate-2 on bloodundifferentiated cells, CD34 positive cells were cultured in aserum-free semi solid medium in the presence of EXS2Fc and knowncytokines, and the number of colony forming cells were observed.

CD34 positive cells of human umbilical cord blood or human normal bonemarrow blood were isolated from the mononuclear cells, which weretreated by silica solution (Immunological and Biological ResearchInstitute, Japan) according to the attached explanation leaflet andfractionated from the low density cellular fraction (<1.077 g/ml) bydensitometric centrifugation of Ficoll pack (Pharmacia Inc., Sweden).

Separation of CD34 positive cells was performed by using Dynabeads M-45CD34 or DETACH a BEADS CD34 (Dynal Inc., Norway) and according to theattached explanation leaflets. After separation, the purity was measuredas follows. Cells were stained by FITC labeled CD34 antibody HPCA2(Beckton-Deckinson Inc., U.S.A.) and examined by flow-cytometer (FACSCalibur, Beckton-Deckinson., U.S.A.). Purity above 85% was confirmed foruse.

The thus isolated CD34 positive cells were suspended homogeneously toform 400 cells/ml of the medium hereinbelow, and spread in a 35 mm dish(Falcon Inc., U.S.A.), then cultured for 2 weeks in a carbon dioxideincubator at 37° C. under 5% carbon dioxide, 5% oxygen, 90% nitrogen and100% humidity. The formed blood colonies were counted under an invertmicroscope.

A medium used is α-medium (GIBCO-BRL Inc., U.S.A.), containing 2%deionized bovine serum albumin (BSA, Sigma, U.S.A.), 10 μg/ml humaninsulin (Sigma, U.S.A.), 200 μg/ml transferring (Sigma, U.S.A.), 10-5 M2-mercaptoethanol (Nakarai Tesk Co., Japan), 160 μg/ml soy-bean lecithin(Sigma, U.S.A.), 96 μg/ml cholesterol (Sigma, U.S.A.) and 0.9%methylcellulose (Wako Pure Chemicals, Japan).

To the above medium, under the following conditions of cytokines, humanSerrate-2 extracellular Ig chimera protein (EXS2Fc) was added to thefinal concentration of 1 μg/ml. For control, human IgG1 (Athens Researchand Technology Inc., U.S.A.) was added with the same concentration inorder to observe the effect of IgGFc region.

Conditions of cytokines are as follows. 100 ng/ml, human SCF, 10 ng/mlhuman IL-3, 100 ng/ml human IL-6, 2U/ml Epo (Chugai Seiyaku Co., Japan)and 10 ng/ml human G-CSF (Chugai Seiyaku Co., Japan).

Results are shown in Table 1. Number of colonies/400 CD34+ cells areshown in mean of n=3. Four different origin human umbilical cord bloodCD34 positive cells were used.

TABLE 1 EXS2Fc not added EXS2Fc added Experiment 1 30.1  12.8 Experiment2 48.3. 40.7 Experiment 3 38.2. 28.1 Experiment 4 50.9. 37.1

As shown in Table 1, the human Serrate-2 has an action against fourdifferent origin umbilical cord blood CD34 positive cells. Therefore,human Serrate-2 of the present invention has suppressive action fordifferentiation of blood undifferentiated cells including blood cells.

Example 9

Effect of Human Serrate-2 on Blood Undifferentiated Cell LTC-IC inLiquid Culture

In order to observe physiological action of human Serrate-2 on the bloodundifferentiated cells, umbilical cord blood positive cells werecultured for two weeks in the serum-free liquid medium in the presenceof EXS2Fc and known cytokines, and the numbers of LTC-IC, which wasthought to represent most undifferentiated cells at present, wereobserved.

The umbilical cord blood mononuclear CD34 positive cells, 100000 to20000 cells, separated by a method described in Example 8 were culturedin the following medium for 2 weeks. Numbers of LTC-IC in 3 experimentalgroups, which include a group before cultivation, a group of EXS2Fc anda control group, were determined.

Media used in liquid culture medium were α-medium with 2% BSA addedthereto, 10 μg/ml human insulin, 200 μg/ml transferring, 40 μg/ml lowdensity lipoprotein, 10-5 M 2-mercaptoethanol, further containing 100ng/ml human SCF, 10 ng/ml human IL-3, and 100 ng/ml IL-6. EXS2Fc 1 μg/mlwas added 1 the above medium. In the control group, human IgG1 was addedin the equal concentration.

Preparation of human bone marrow stromal cell layer used for LTC-IC, andquantitative assay of frequency of LTC-IC by a limit dilution wereperformed according to a method of Sutherland et al. (Blood, 74, 1563-,1989 and Proc. Natl. Acad. Sci. USA, 87, 3584-, 1990).

The bone marrow mononuclear cells, 1-2×10 7 cells, obtained in Example 8before the separation and without the liquid silica treatment, werecultured in LTC medium (MyceloCult, Stem Cell Technologies Inc., Canada)5 ml containing hydrocortisone 1 μM (Upjohn Japan Co., Japan) in T-25flask (Falcon Inc., U.S.A.) at 37° C. under 5% carbon dioxide and 100%humidity in the carbon dioxide incubator. Culture was conducted untilthe adhesive cell layers of the stromal cell formation spread more than80% of the bottom area of the culture flask. Detachment of the celllayer was performed by treating with EDTA solution (Cosmobio Co.,Japan). Cells were plated in the 96 well plate (Beckton-Deckinson Inc.,U.S.A.), about 2×10 4 cells/well and recultivation was continued in thesame medium. X-ray, 15 Gy, 250 KV peak was irradiated to thereconstituted stromal cell layer. Growth of stromal cells was stoppedand blood cells in the stromal cells were removed. The thus preparedstromal cells were used as stromal cell layer for the experiments.

In the assay of LTC-IC, cell counts in each group were adjusted withinthe ranges of 25-400 cells/well for CD34 positive cells before thecultivation, and 625-20000 cells/well for the cells after thecultivation, and cells were diluted for a six step-dilution within theseranges. Each dilution step of cells was cocultured with the abovestromal cell layer in the 96 well plate, for 16 wells of one dilutionstep. Culture was performed in the same medium as used in stromalformation, at 37° C., 5% carbon dioxide and 100% humidity in the carbondioxide gas incubator for 5 weeks. Cells in suspension and in attachmentafter cultivation were recovered in each well. Collected cells weretransferred to the semi-solid culture medium consisting of α-mediumcontaining 0.9% methylcellulose, 30% fetal calf serum (FCS, ICNBiochemical Japan), 1% BSA, 10-5 M 2-mercaptoethanol, 100 ng/ml humanSCF, 10 ng/ml human IL-3, 100 ng/ml human IL-6, 2 U/ml Epo and 10 ng/mlhuman G-CSF. After 2 weeks of cultivation, colony forming cells weredetected in the same way as described in Example 8, and numbers of wellsin which colony forming cells were found, were detected. Incidence ofLTC-IC was calculated according to the method of Taswell et al. (J.Immunol. 126, 1614-, 1981) based on the above data. Results are shown inTable 2.

TABLE 2 Before EXS2Fc EXS2Fc cultivation not added added Total number ofcells 19475 1210000 930000 Number of LTC-IC 185 23 5

From the results shown in Table 2, human Serrate-2 has an action againstLTC-IC and reduces its number.

Example 10

Effect of Human Serrate-2 on Growth of Vascular Endothelial Cells

The vascular endothelial cells used were passage cultures of fourgenerations of normal human aortic endothelial cells and normal humanpulmonary arterial endothelial cells (Kurabo Inc., Japan). Cells wereplated 500 cells/well in 96 well plate for tissue culture (Falcon Inc.,U.S.A.) in the tertiary passage culture, and cultured in a medium withlow serum level for growth of vascular endothelial cells (HuMedia-EG2,Kurabo Inc., Japan) containing human recombinant EGF (KuraboInc., Japan)100 ng/ml and human recombinant EGF-B 5 ng/ml. Human Serrate-2extracellular chimera protein (EXS2Fc) was added to the finalconcentration of 1 μg/ml. For control, human IgG1 (Athens Research andTechnology Inc., U.S.A.) was added with the same concentration in orderto observe effect of IgGFc region. A control experiment was conductedwithout adding protein except for HuMedia-EG2. Culture was performed at37° C., under 5% carbon dioxide and 100% humidity for 3 days and thenumber of cells was calculated.

Vascular endothelial cell counts were performed by using NR reagent set(Kurabo Inc., Japan). The method was developed by Borenfeund and Puerner(Journal of Tissue Culture Methods, 9(1), 7-9, 1984), i.e. the neutralred method which applied that vital stain pigment neutral red(3-amino-7-dimetylamino-2-methylphenazine hydrochloride) passed throughplasma membrane of living cells and was accumulated in lysosome.

Absorption at 540 nm was measured by using immuno reader (NJ-2000, JapanIntermed Inc., Japan).

Results showed that in case of aortic endothelial cells, absorption inthe control group was at an optical density (OD) 0.21±0.02, which isalmost the same level of human IgG1 added group 0.20±0.01, and in EXS2Fccontaining group, it was 0.10±0.02 which was significantly smaller thanthe control.

In case of pulmonary arterial endothelial cells, the control groupshowed 0.15±0.01 and the human IgG1 containing group showed almost samelevel 0.16±0.02, whereas EXS2Fc added group shows significantly lowlevel of 0.07±0.02. This result indicated that EXS2Fc suppresses growthof vascular endothelial cells.

Human Serrate-2 of the present invention therefore has an action forregulating differentiation of undifferentiated cells, and can be used asa novel regulating agent for differentiation of cells.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 32 <210> SEQ ID NO 1 <211>LENGTH: 214 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:1 Met Gly Tyr Phe Glu Leu Gln Leu Ser Ala Leu Arg Asn Val Asn Gly 1 5 1015 Glu Leu Leu Ser Gly Ala Cys Cys Asp Gly Asp Gly Arg Thr Thr Arg 20 2530 Ala Gly Gly Cys Gly His Asp Glu Cys Asp Thr Tyr Val Arg Val Cys 35 4045 Leu Lys Glu Tyr Gln Ala Lys Val Thr Pro Thr Gly Pro Cys Ser Tyr 50 5560 Gly His Gly Ala Thr Pro Val Leu Gly Gly Asn Ser Phe Tyr Leu Pro 65 7075 80 Pro Ala Gly Ala Ala Gly Asp Arg Ala Arg Ala Arg Ala Arg Ala Gly 8590 95 Gly Asp Gln Asp Pro Gly Leu Val Val Ile Pro Phe Gln Phe Ala Trp100 105 110 Pro Arg Ser Phe Thr Leu Ile Val Glu Ala Trp Asp Trp Asp AsnAsp 115 120 125 Thr Thr Pro Asn Glu Glu Leu Leu Ile Glu Arg Val Ser HisAla Gly 130 135 140 Met Ile Asn Pro Glu Asp Arg Trp Lys Ser Leu His PheSer Gly His 145 150 155 160 Val Ala His Leu Glu Leu Gln Ile Arg Val ArgCys Asp Glu Asn Tyr 165 170 175 Tyr Ser Ala Thr Cys Asn Lys Phe Cys ArgPro Arg Asn Asp Phe Phe 180 185 190 Gly His Tyr Thr Cys Asp Gln Tyr GlyAsn Lys Ala Cys Met Asp Gly 195 200 205 Trp Met Gly Lys Glu Cys 210<210> SEQ ID NO 2 <211> LENGTH: 1055 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 2 Met Gly Tyr Phe Glu Leu Gln Leu Ser AlaLeu Arg Asn Val Asn Gly 1 5 10 15 Glu Leu Leu Ser Gly Ala Cys Cys AspGly Asp Gly Arg Thr Thr Arg 20 25 30 Ala Gly Gly Cys Gly His Asp Glu CysAsp Thr Tyr Val Arg Val Cys 35 40 45 Leu Lys Glu Tyr Gln Ala Lys Val ThrPro Thr Gly Pro Cys Ser Tyr 50 55 60 Gly His Gly Ala Thr Pro Val Leu GlyGly Asn Ser Phe Tyr Leu Pro 65 70 75 80 Pro Ala Gly Ala Ala Gly Asp ArgAla Arg Ala Arg Ala Arg Ala Gly 85 90 95 Gly Asp Gln Asp Pro Gly Leu ValVal Ile Pro Phe Gln Phe Ala Trp 100 105 110 Pro Arg Ser Phe Thr Leu IleVal Glu Ala Trp Asp Trp Asp Asn Asp 115 120 125 Thr Thr Pro Asn Glu GluLeu Leu Ile Glu Arg Val Ser His Ala Gly 130 135 140 Met Ile Asn Pro GluAsp Arg Trp Lys Ser Leu His Phe Ser Gly His 145 150 155 160 Val Ala HisLeu Glu Leu Gln Ile Arg Val Arg Cys Asp Glu Asn Tyr 165 170 175 Tyr SerAla Thr Cys Asn Lys Phe Cys Arg Pro Arg Asn Asp Phe Phe 180 185 190 GlyHis Tyr Thr Cys Asp Gln Tyr Gly Asn Lys Ala Cys Met Asp Gly 195 200 205Trp Met Gly Lys Glu Cys Lys Glu Ala Val Cys Lys Gln Gly Cys Asn 210 215220 Leu Leu His Gly Gly Cys Thr Val Pro Gly Glu Cys Arg Cys Ser Tyr 225230 235 240 Gly Trp Gln Gly Arg Phe Cys Asp Glu Cys Val Pro Tyr Pro GlyCys 245 250 255 Val His Gly Ser Cys Val Glu Pro Trp Gln Cys Asn Cys GluThr Asn 260 265 270 Trp Gly Gly Leu Leu Cys Asp Lys Asp Leu Asn Tyr CysGlu Ser His 275 280 285 His Pro Cys Thr Asn Gly Gly Thr Cys Ile Asn AlaGlu Pro Asp Gln 290 295 300 Tyr Arg Cys Thr Cys Pro Asp Gly Tyr Ser GlyArg Asn Cys Glu Lys 305 310 315 320 Ala Glu His Ala Cys Thr Ser Asn ProCys Ala Asn Gly Gly Ser Cys 325 330 335 His Glu Val Pro Ser Gly Phe GluCys His Cys Pro Ser Gly Trp Ser 340 345 350 Gly Pro Thr Cys Ala Leu AspIle Asp Glu Cys Ala Ser Asn Pro Cys 355 360 365 Ala Ala Gly Gly Thr CysVal Asp Gln Val Asp Gly Phe Glu Cys Ile 370 375 380 Cys Pro Glu Gln TrpVal Gly Ala Thr Cys Gln Leu Asp Ala Asn Glu 385 390 395 400 Cys Glu GlyLys Pro Cys Leu Asn Ala Phe Ser Cys Lys Asn Leu Ile 405 410 415 Gly GlyTyr Tyr Cys Asp Cys Ile Pro Gly Trp Lys Gly Ile Asn Cys 420 425 430 HisIle Asn Val Asn Asp Cys Arg Gly Gln Cys Gln His Gly Gly Thr 435 440 445Cys Lys Asp Leu Val Asn Gly Tyr Gln Cys Val Cys Pro Arg Gly Phe 450 455460 Gly Gly Arg His Cys Glu Leu Glu Arg Asp Lys Cys Ala Ser Ser Pro 465470 475 480 Cys His Ser Gly Gly Leu Cys Glu Asp Leu Ala Asp Gly Phe HisCys 485 490 495 His Cys Pro Gln Gly Phe Ser Gly Pro Leu Cys Glu Val AspVal Asp 500 505 510 Leu Cys Glu Pro Ser Pro Cys Arg Asn Gly Ala Arg CysTyr Asn Leu 515 520 525 Glu Gly Asp Tyr Tyr Cys Ala Cys Pro Asp Asp PheGly Gly Lys Asn 530 535 540 Cys Ser Val Pro Arg Glu Pro Cys Pro Gly GlyAla Cys Arg Val Ile 545 550 555 560 Asp Gly Cys Gly Ser Asp Ala Gly ProGly Met Pro Gly Thr Ala Ala 565 570 575 Ser Gly Val Cys Gly Pro His GlyArg Cys Val Ser Gln Pro Gly Gly 580 585 590 Asn Phe Ser Cys Ile Cys AspSer Gly Phe Thr Gly Thr Tyr Cys His 595 600 605 Glu Asn Ile Asp Asp CysLeu Gly Gln Pro Cys Arg Asn Gly Gly Thr 610 615 620 Cys Ile Asp Glu ValAsp Ala Phe Arg Cys Phe Cys Pro Ser Gly Trp 625 630 635 640 Glu Gly GluLeu Cys Asp Thr Asn Pro Asn Asp Cys Leu Pro Asp Pro 645 650 655 Cys HisSer Arg Gly Arg Cys Tyr Asp Leu Val Asn Asp Phe Tyr Cys 660 665 670 AlaCys Asp Asp Gly Trp Lys Gly Lys Thr Cys His Ser Arg Glu Phe 675 680 685Gln Cys Asp Ala Tyr Thr Cys Ser Asn Gly Gly Thr Cys Tyr Asp Ser 690 695700 Gly Asp Thr Phe Arg Cys Ala Cys Pro Pro Gly Trp Lys Gly Ser Thr 705710 715 720 Cys Ala Val Ala Lys Asn Ser Ser Cys Leu Pro Asn Pro Cys ValAsn 725 730 735 Gly Gly Thr Cys Val Gly Ser Gly Ala Ser Phe Ser Cys IleCys Arg 740 745 750 Asp Gly Trp Glu Gly Arg Thr Cys Thr His Asn Thr AsnAsp Cys Asn 755 760 765 Pro Leu Pro Cys Tyr Asn Gly Gly Ile Cys Val AspGly Val Asn Trp 770 775 780 Phe Arg Cys Glu Cys Ala Pro Gly Phe Ala GlyPro Asp Cys Arg Ile 785 790 795 800 Asn Ile Asp Glu Cys Gln Ser Ser ProCys Ala Tyr Gly Ala Thr Cys 805 810 815 Val Asp Glu Ile Asn Gly Tyr ArgCys Ser Cys Pro Pro Gly Arg Ala 820 825 830 Gly Pro Arg Cys Gln Glu ValIle Gly Phe Gly Arg Ser Cys Trp Ser 835 840 845 Arg Gly Thr Pro Phe ProHis Gly Ser Ser Trp Val Glu Asp Cys Asn 850 855 860 Ser Cys Arg Cys LeuAsp Gly Arg Arg Asp Cys Ser Lys Val Trp Cys 865 870 875 880 Gly Trp LysPro Cys Leu Leu Ala Gly Gln Pro Glu Ala Leu Ser Ala 885 890 895 Gln CysPro Leu Gly Gln Arg Cys Leu Glu Lys Ala Pro Gly Gln Cys 900 905 910 LeuArg Pro Pro Cys Glu Ala Trp Gly Glu Cys Gly Ala Glu Glu Pro 915 920 925Pro Ser Thr Pro Cys Leu Pro Arg Ser Gly His Leu Asp Asn Asn Cys 930 935940 Ala Arg Leu Thr Leu His Phe Asn Arg Asp His Val Pro Gln Gly Thr 945950 955 960 Thr Val Gly Ala Ile Cys Ser Gly Ile Arg Ser Leu Pro Ala ThrArg 965 970 975 Ala Val Ala Arg Asp Arg Leu Leu Val Leu Leu Cys Asp ArgAla Ser 980 985 990 Ser Gly Ala Ser Ala Val Glu Val Ala Val Ser Phe SerPro Ala Arg 995 1000 1005 Asp Leu Pro Asp Ser Ser Leu Ile Gln Gly AlaAla His Ala Ile Val 1010 1015 1020 Ala Ala Ile Thr Gln Arg Gly Asn SerSer Leu Leu Leu Ala Val Thr 1025 1030 1035 1040 Glu Val Lys Val Glu ThrVal Val Thr Gly Gly Ser Ser Thr Gly 1045 1050 1055 <210> SEQ ID NO 3<211> LENGTH: 1212 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 3 Met Gly Tyr Phe Glu Leu Gln Leu Ser Ala Leu Arg Asn Val AsnGly 1 5 10 15 Glu Leu Leu Ser Gly Ala Cys Cys Asp Gly Asp Gly Arg ThrThr Arg 20 25 30 Ala Gly Gly Cys Gly His Asp Glu Cys Asp Thr Tyr Val ArgVal Cys 35 40 45 Leu Lys Glu Tyr Gln Ala Lys Val Thr Pro Thr Gly Pro CysSer Tyr 50 55 60 Gly His Gly Ala Thr Pro Val Leu Gly Gly Asn Ser Phe TyrLeu Pro 65 70 75 80 Pro Ala Gly Ala Ala Gly Asp Arg Ala Arg Ala Arg AlaArg Ala Gly 85 90 95 Gly Asp Gln Asp Pro Gly Leu Val Val Ile Pro Phe GlnPhe Ala Trp 100 105 110 Pro Arg Ser Phe Thr Leu Ile Val Glu Ala Trp AspTrp Asp Asn Asp 115 120 125 Thr Thr Pro Asn Glu Glu Leu Leu Ile Glu ArgVal Ser His Ala Gly 130 135 140 Met Ile Asn Pro Glu Asp Arg Trp Lys SerLeu His Phe Ser Gly His 145 150 155 160 Val Ala His Leu Glu Leu Gln IleArg Val Arg Cys Asp Glu Asn Tyr 165 170 175 Tyr Ser Ala Thr Cys Asn LysPhe Cys Arg Pro Arg Asn Asp Phe Phe 180 185 190 Gly His Tyr Thr Cys AspGln Tyr Gly Asn Lys Ala Cys Met Asp Gly 195 200 205 Trp Met Gly Lys GluCys Lys Glu Ala Val Cys Lys Gln Gly Cys Asn 210 215 220 Leu Leu His GlyGly Cys Thr Val Pro Gly Glu Cys Arg Cys Ser Tyr 225 230 235 240 Gly TrpGln Gly Arg Phe Cys Asp Glu Cys Val Pro Tyr Pro Gly Cys 245 250 255 ValHis Gly Ser Cys Val Glu Pro Trp Gln Cys Asn Cys Glu Thr Asn 260 265 270Trp Gly Gly Leu Leu Cys Asp Lys Asp Leu Asn Tyr Cys Gly Ser His 275 280285 His Pro Cys Thr Asn Gly Gly Thr Cys Ile Asn Ala Glu Pro Asp Gln 290295 300 Tyr Arg Cys Thr Cys Pro Asp Gly Tyr Ser Gly Arg Asn Cys Glu Lys305 310 315 320 Ala Glu His Ala Cys Thr Ser Asn Pro Cys Ala Asn Gly GlySer Cys 325 330 335 His Glu Val Pro Ser Gly Phe Glu Cys His Cys Pro SerGly Trp Ser 340 345 350 Gly Pro Thr Cys Ala Leu Asp Ile Asp Glu Cys AlaSer Asn Pro Cys 355 360 365 Ala Ala Gly Gly Thr Cys Val Asp Gln Val AspGly Phe Glu Cys Ile 370 375 380 Cys Pro Glu Gln Trp Val Gly Ala Thr CysGln Leu Asp Ala Asn Glu 385 390 395 400 Cys Glu Gly Lys Pro Cys Leu AsnAla Phe Ser Cys Lys Asn Leu Ile 405 410 415 Gly Gly Tyr Tyr Cys Asp CysIle Pro Gly Trp Lys Gly Ile Asn Cys 420 425 430 His Ile Asn Val Asn AspCys Arg Gly Gln Cys Gln His Gly Gly Thr 435 440 445 Cys Lys Asp Leu ValAsn Gly Tyr Gln Cys Val Cys Pro Arg Gly Phe 450 455 460 Gly Gly Arg HisCys Glu Leu Glu Arg Asp Lys Cys Ala Ser Ser Pro 465 470 475 480 Cys HisSer Gly Gly Leu Cys Glu Asp Leu Ala Asp Gly Phe His Cys 485 490 495 HisCys Pro Gln Gly Phe Ser Gly Pro Leu Cys Glu Val Asp Val Asp 500 505 510Leu Cys Glu Pro Ser Pro Cys Arg Asn Gly Ala Arg Cys Tyr Asn Leu 515 520525 Glu Gly Asp Tyr Tyr Cys Ala Cys Pro Asp Asp Phe Gly Gly Lys Asn 530535 540 Cys Ser Val Pro Arg Glu Pro Cys Pro Gly Gly Ala Cys Arg Val Ile545 550 555 560 Asp Gly Cys Gly Ser Asp Ala Gly Pro Gly Met Pro Gly ThrAla Ala 565 570 575 Ser Gly Val Cys Gly Pro His Gly Arg Cys Val Ser GlnPro Gly Gly 580 585 590 Asn Phe Ser Cys Ile Cys Asp Ser Gly Phe Thr GlyThr Tyr Cys His 595 600 605 Glu Asn Ile Asp Asp Cys Leu Gly Gln Pro CysArg Asn Gly Gly Thr 610 615 620 Cys Ile Asp Glu Val Asp Ala Phe Arg CysPhe Cys Pro Ser Gly Trp 625 630 635 640 Glu Gly Glu Leu Cys Asp Thr AsnPro Asn Asp Cys Leu Pro Asp Pro 645 650 655 Cys His Ser Arg Gly Arg CysTyr Asp Leu Val Asn Asp Phe Tyr Cys 660 665 670 Ala Cys Asp Asp Gly TrpLys Gly Lys Thr Cys His Ser Arg Glu Phe 675 680 685 Gln Cys Asp Ala TyrThr Cys Ser Asn Gly Gly Thr Cys Tyr Asp Ser 690 695 700 Gly Asp Thr PheArg Cys Ala Cys Pro Pro Gly Trp Lys Gly Ser Thr 705 710 715 720 Cys AlaVal Ala Lys Asn Ser Ser Cys Leu Pro Asn Pro Cys Val Asn 725 730 735 GlyGly Thr Cys Val Gly Ser Gly Ala Ser Phe Ser Cys Ile Cys Arg 740 745 750Asp Gly Trp Glu Gly Arg Thr Cys Thr His Asn Thr Asn Asp Cys Asn 755 760765 Pro Leu Pro Cys Tyr Asn Gly Gly Ile Cys Val Asp Gly Val Asn Trp 770775 780 Phe Arg Cys Glu Cys Ala Pro Gly Phe Ala Gly Pro Asp Cys Arg Ile785 790 795 800 Asn Ile Asp Glu Cys Gln Ser Ser Pro Cys Ala Tyr Gly AlaThr Cys 805 810 815 Val Asp Glu Ile Asn Gly Tyr Arg Cys Ser Cys Pro ProGly Arg Ala 820 825 830 Gly Pro Arg Cys Gln Glu Val Ile Gly Phe Gly ArgSer Cys Trp Ser 835 840 845 Arg Gly Thr Pro Phe Pro His Gly Ser Ser TrpVal Glu Asp Cys Asn 850 855 860 Ser Cys Arg Cys Leu Asp Gly Arg Arg AspCys Ser Lys Val Trp Cys 865 870 875 880 Gly Trp Lys Pro Cys Leu Leu AlaGly Gln Pro Glu Ala Leu Ser Ala 885 890 895 Gln Cys Pro Leu Gly Gln ArgCys Leu Glu Lys Ala Pro Gly Gln Cys 900 905 910 Leu Arg Pro Pro Cys GluAla Trp Gly Glu Cys Gly Ala Glu Glu Pro 915 920 925 Pro Ser Thr Pro CysLeu Pro Arg Ser Gly His Leu Asp Asn Asn Cys 930 935 940 Ala Arg Leu ThrLeu His Phe Asn Arg Asp His Val Pro Gln Gly Thr 945 950 955 960 Thr ValGly Ala Ile Cys Ser Gly Ile Arg Ser Leu Pro Ala Thr Arg 965 970 975 AlaVal Ala Arg Asp Arg Leu Leu Val Leu Leu Cys Asp Arg Ala Ser 980 985 990Ser Gly Ala Ser Ala Val Glu Val Ala Val Ser Phe Ser Pro Ala Arg 995 10001005 Asp Leu Pro Asp Ser Ser Leu Ile Gln Gly Ala Ala His Ala Ile Val1010 1015 1020 Ala Ala Ile Thr Gln Arg Gly Asn Ser Ser Leu Leu Leu AlaVal Thr 1025 1030 1035 1040 Glu Val Lys Val Glu Thr Val Val Thr Gly GlySer Ser Thr Gly Leu 1045 1050 1055 Leu Val Pro Val Leu Cys Gly Ala PheSer Val Leu Trp Leu Ala Cys 1060 1065 1070 Val Val Leu Cys Val Trp TrpThr Arg Lys Arg Arg Lys Glu Arg Glu 1075 1080 1085 Arg Ser Arg Leu ProArg Glu Glu Ser Ala Asn Asn Gln Trp Ala Pro 1090 1095 1100 Leu Asn ProIle Arg Asn Pro Ile Glu Arg Pro Gly Gly His Lys Asp 1105 1110 1115 1120Val Leu Tyr Gln Cys Lys Asn Phe Thr Pro Pro Pro Arg Arg Ala Asp 11251130 1135 Glu Ala Leu Pro Gly Pro Ala Gly His Ala Ala Val Arg Glu AspGlu 1140 1145 1150 Glu Asp Glu Asp Leu Gly Arg Gly Glu Glu Asp Ser LeuGlu Ala Glu 1155 1160 1165 Lys Phe Leu Ser His Lys Phe Thr Lys Asp ProGly Arg Ser Pro Gly 1170 1175 1180 Arg Pro Ala His Trp Ala Ser Gly ProLys Val Asp Asn Arg Ala Val 1185 1190 1195 1200 Arg Ser Ile Asn Glu AlaArg Tyr Ala Gly Lys Glu 1205 1210 <210> SEQ ID NO 4 <211> LENGTH: 3955<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (12)..(3725) <221> NAME/KEY: sig_peptide<222> LOCATION: (12)..(89) <221> NAME/KEY: mat_peptide <222> LOCATION:(90)..(3725) <400> SEQUENCE: 4 tcgcgggggc a atg cgg gcg cag ggc cgg gggcgc ctt ccc cgg cgg ctg 50 Met Arg Ala Gln Gly Arg Gly Arg Leu Pro ArgArg Leu -25 -20 -15 ctg ctg ctg ctg gcg ctc tgg gtg cag gcg gcg cgg cccatg ggc tat 98 Leu Leu Leu Leu Ala Leu Trp Val Gln Ala Ala Arg Pro MetGly Tyr -10 -5 -1 1 ttc gag ctg cag ctg agc gcg ctg cgg aac gtg aac ggggag ctg ctg 146 Phe Glu Leu Gln Leu Ser Ala Leu Arg Asn Val Asn Gly GluLeu Leu 5 10 15 agc ggc gcc tgc tgt gac ggc gac ggc cgg aca acg cgc gcgggg ggc 194 Ser Gly Ala Cys Cys Asp Gly Asp Gly Arg Thr Thr Arg Ala GlyGly 20 25 30 35 tgc ggc cac gac gag tgc gac acg tac gtg cgc gtg tgc cttaag gag 242 Cys Gly His Asp Glu Cys Asp Thr Tyr Val Arg Val Cys Leu LysGlu 40 45 50 tac cag gcc aag gtg acg ccc acg ggg ccc tgc agc tac ggc cacggc 290 Tyr Gln Ala Lys Val Thr Pro Thr Gly Pro Cys Ser Tyr Gly His Gly55 60 65 gcc acg ccc gtg ctg ggc ggc aac tcc ttc tac ctg ccg ccg gcg ggc338 Ala Thr Pro Val Leu Gly Gly Asn Ser Phe Tyr Leu Pro Pro Ala Gly 7075 80 gct gcg ggg gac cga gcg cgg gcg cgg gcc cgg gcc ggc ggc gac cag386 Ala Ala Gly Asp Arg Ala Arg Ala Arg Ala Arg Ala Gly Gly Asp Gln 8590 95 gac ccg ggc ctc gtc gtc atc ccc ttc cag ttc gcc tgg ccg cgc tcc434 Asp Pro Gly Leu Val Val Ile Pro Phe Gln Phe Ala Trp Pro Arg Ser 100105 110 115 ttt acc ctc atc gtg gag gcc tgg gac tgg gac aac gat acc accccg 482 Phe Thr Leu Ile Val Glu Ala Trp Asp Trp Asp Asn Asp Thr Thr Pro120 125 130 aat gag gag ctg ctg atc gag cga gtg tcg cat gcc ggc atg atcaac 530 Asn Glu Glu Leu Leu Ile Glu Arg Val Ser His Ala Gly Met Ile Asn135 140 145 ccg gag gac cgc tgg aag agc ctg cac ttc agc ggc cac gtg gcgcac 578 Pro Glu Asp Arg Trp Lys Ser Leu His Phe Ser Gly His Val Ala His150 155 160 ctg gag ctg cag atc cgc gtg cgc tgc gac gag aac tac tac agcgcc 626 Leu Glu Leu Gln Ile Arg Val Arg Cys Asp Glu Asn Tyr Tyr Ser Ala165 170 175 act tgc aac aag ttc tgc cgg ccc cgc aac gac ttt ttc ggc cactac 674 Thr Cys Asn Lys Phe Cys Arg Pro Arg Asn Asp Phe Phe Gly His Tyr180 185 190 195 acc tgc gac cag tac ggc aac aag gcc tgc atg gac ggc tggatg ggc 722 Thr Cys Asp Gln Tyr Gly Asn Lys Ala Cys Met Asp Gly Trp MetGly 200 205 210 aag gag tgc aag gaa gct gtg tgt aaa caa ggg tgt aat ttgctc cac 770 Lys Glu Cys Lys Glu Ala Val Cys Lys Gln Gly Cys Asn Leu LeuHis 215 220 225 ggg gga tgc acc gtg cct ggg gag tgc agg tgc agc tac ggctgg caa 818 Gly Gly Cys Thr Val Pro Gly Glu Cys Arg Cys Ser Tyr Gly TrpGln 230 235 240 ggg agg ttc tgc gat gag tgt gtc ccc tac ccc ggc tgc gtgcat ggc 866 Gly Arg Phe Cys Asp Glu Cys Val Pro Tyr Pro Gly Cys Val HisGly 245 250 255 agt tgt gtg gag ccc tgg cag tgc aac tgt gag acc aac tggggc ggc 914 Ser Cys Val Glu Pro Trp Gln Cys Asn Cys Glu Thr Asn Trp GlyGly 260 265 270 275 ctg ctc tgt gac aaa gac ctg aac tac tgt ggc agc caccac ccc tgc 962 Leu Leu Cys Asp Lys Asp Leu Asn Tyr Cys Gly Ser His HisPro Cys 280 285 290 acc aac gga ggc acg tgc atg aac gcc gag cct gac cagtac cgc tgc 1010 Thr Asn Gly Gly Thr Cys Met Asn Ala Glu Pro Asp Gln TyrArg Cys 295 300 305 acc tgc cct gac ggc tac tcg ggc agg aac tgt gag aaggct gag cac 1058 Thr Cys Pro Asp Gly Tyr Ser Gly Arg Asn Cys Glu Lys AlaGlu His 310 315 320 gcc tgc acc tcc aac ccg tgt gcc aac ggg ggc tct tgccat gag gtg 1106 Ala Cys Thr Ser Asn Pro Cys Ala Asn Gly Gly Ser Cys HisGlu Val 325 330 335 ccg tcc ggc ttc gaa tgc cac tgc cca tcg ggc tgg agcggg ccc acc 1154 Pro Ser Gly Phe Glu Cys His Cys Pro Ser Gly Trp Ser GlyPro Thr 340 345 350 355 tgt gcc ctt gac atc gat gag tgt gct tcg aac ccgtgt gcg gcc ggt 1202 Cys Ala Leu Asp Ile Asp Glu Cys Ala Ser Asn Pro CysAla Ala Gly 360 365 370 ggc acc tgt gtg gac cag gtg gac ggc ttt gag tgcatc tgc ccc gag 1250 Gly Thr Cys Val Asp Gln Val Asp Gly Phe Glu Cys IleCys Pro Glu 375 380 385 cag tgg gtg ggg gcc acc tgc cag ctg gac gcc aatgag tgt gaa ggg 1298 Gln Trp Val Gly Ala Thr Cys Gln Leu Asp Ala Asn GluCys Glu Gly 390 395 400 aag cca tgc ctt aac gct ttt tct tgc aaa aac ctgatt ggc ggc tat 1346 Lys Pro Cys Leu Asn Ala Phe Ser Cys Lys Asn Leu IleGly Gly Tyr 405 410 415 tac tgt gat tgc atc ccg ggc tgg aag ggc atc aactgc cat atc aac 1394 Tyr Cys Asp Cys Ile Pro Gly Trp Lys Gly Ile Asn CysHis Ile Asn 420 425 430 435 gtc aac gac tgt cgc ggg cag tgt cag cat gggggc acc tgc aag gac 1442 Val Asn Asp Cys Arg Gly Gln Cys Gln His Gly GlyThr Cys Lys Asp 440 445 450 ctg gtg aac ggg tac cag tgt gtg tgc cca cggggc ttc gga ggc cgg 1490 Leu Val Asn Gly Tyr Gln Cys Val Cys Pro Arg GlyPhe Gly Gly Arg 455 460 465 cat tgc gag ctg gaa cga gac aag tgt gcc agcagc ccc tgc cac agc 1538 His Cys Glu Leu Glu Arg Asp Lys Cys Ala Ser SerPro Cys His Ser 470 475 480 ggc ggc ctc tgc gag gac ctg gcc gac ggc ttccac tgc cac tgc ccc 1586 Gly Gly Leu Cys Glu Asp Leu Ala Asp Gly Phe HisCys His Cys Pro 485 490 495 cag ggc ttc tcc ggg cct ctc tgt gag gtg gatgtc gac ctt tgt gag 1634 Gln Gly Phe Ser Gly Pro Leu Cys Glu Val Asp ValAsp Leu Cys Glu 500 505 510 515 cca agc ccc tgc cgg aac ggc gct cgc tgctat aac ctg gag ggt gac 1682 Pro Ser Pro Cys Arg Asn Gly Ala Arg Cys TyrAsn Leu Glu Gly Asp 520 525 530 tat tac tgc gcc tgc cct gat gac ttt ggtggc aag aac tgc tcc gtg 1730 Tyr Tyr Cys Ala Cys Pro Asp Asp Phe Gly GlyLys Asn Cys Ser Val 535 540 545 ccc cgc gag ccg tgc cct ggc ggg gcc tgcaga gtg atc gat ggc tgc 1778 Pro Arg Glu Pro Cys Pro Gly Gly Ala Cys ArgVal Ile Asp Gly Cys 550 555 560 ggg tca gac gcg ggg cct ggg atg cct ggcaca gca gcc tcc ggc gtg 1826 Gly Ser Asp Ala Gly Pro Gly Met Pro Gly ThrAla Ala Ser Gly Val 565 570 575 tgt ggc ccc cat gga cgc tgc gtc agc cagcca ggg ggc aac ttt tcc 1874 Cys Gly Pro His Gly Arg Cys Val Ser Gln ProGly Gly Asn Phe Ser 580 585 590 595 tgc atc tgt gac agt ggc ttt act ggcacc tac tgc cat gag aac att 1922 Cys Ile Cys Asp Ser Gly Phe Thr Gly ThrTyr Cys His Glu Asn Ile 600 605 610 gac gac tgc ctg ggc cag ccc tgc cgcaat ggg ggc aca tgc atc gat 1970 Asp Asp Cys Leu Gly Gln Pro Cys Arg AsnGly Gly Thr Cys Ile Asp 615 620 625 gag gtg gac gcc ttc cgc tgc ttc tgcccc agc ggc tgg gag ggc gag 2018 Glu Val Asp Ala Phe Arg Cys Phe Cys ProSer Gly Trp Glu Gly Glu 630 635 640 ctc tgc gac acc aat ccc aac gac tgcctt ccc gat ccc tgc cac agc 2066 Leu Cys Asp Thr Asn Pro Asn Asp Cys LeuPro Asp Pro Cys His Ser 645 650 655 cgc ggc cgc tgc tac gac ctg gtc aatgac ttc tac tgt gcg tgc gac 2114 Arg Gly Arg Cys Tyr Asp Leu Val Asn AspPhe Tyr Cys Ala Cys Asp 660 665 670 675 gac ggc tgg aag ggc aag acc tgccac tca cgc gag ttc cag tgc gat 2162 Asp Gly Trp Lys Gly Lys Thr Cys HisSer Arg Glu Phe Gln Cys Asp 680 685 690 gcc tac acc tgc agc aac ggt ggcacc tgc tac gac agc ggc gac acc 2210 Ala Tyr Thr Cys Ser Asn Gly Gly ThrCys Tyr Asp Ser Gly Asp Thr 695 700 705 ttc cgc tgc gcc tgg ccc ccc ggctgg aag ggc agc acc tgc gcc gtc 2258 Phe Arg Cys Ala Trp Pro Pro Gly TrpLys Gly Ser Thr Cys Ala Val 710 715 720 gcc aag aac agc agc tgc ctg cccaac ccc tgt gtg aat ggt ggc acc 2306 Ala Lys Asn Ser Ser Cys Leu Pro AsnPro Cys Val Asn Gly Gly Thr 725 730 735 tgc gtg ggc agc ggg gcc tcc ttctcc tgc atc tgc cgg gac ggc tgg 2354 Cys Val Gly Ser Gly Ala Ser Phe SerCys Ile Cys Arg Asp Gly Trp 740 745 750 755 gag ggt cgt act tgc act cacaat acc aac gac tgc aac cct ctg cct 2402 Glu Gly Arg Thr Cys Thr His AsnThr Asn Asp Cys Asn Pro Leu Pro 760 765 770 tgc tac aat ggt ggc atc tgtgtt gac ggc gtc aac tgg ttc cgc tgc 2450 Cys Tyr Asn Gly Gly Ile Cys ValAsp Gly Val Asn Trp Phe Arg Cys 775 780 785 gag tgt gca cct ggc ttc gcgggg cct gac tgc cgc atc aac atc gac 2498 Glu Cys Ala Pro Gly Phe Ala GlyPro Asp Cys Arg Ile Asn Ile Asp 790 795 800 gag tgc cag tcc tcg ccc tgtgcc tac ggg gcc acg tgt gtg gat gag 2546 Glu Cys Gln Ser Ser Pro Cys AlaTyr Gly Ala Thr Cys Val Asp Glu 805 810 815 atc aac ggg tat cgc tgt agctgc cca ccc ggc cga gcc ggc ccc cgg 2594 Ile Asn Gly Tyr Arg Cys Ser CysPro Pro Gly Arg Ala Gly Pro Arg 820 825 830 835 tgc cag gaa gtg atc gggttc ggg aga tcc tgc tgg tcc cgg ggc act 2642 Cys Gln Glu Val Ile Gly PheGly Arg Ser Cys Trp Ser Arg Gly Thr 840 845 850 ccg ttc cca cac gga agctcc tgg gtg gaa gac tgc aac agc tgc cgc 2690 Pro Phe Pro His Gly Ser SerTrp Val Glu Asp Cys Asn Ser Cys Arg 855 860 865 tgc ctg gat ggc cgc cgtgac tgc agc aag gtg tgg tgc gga tgg aag 2738 Cys Leu Asp Gly Arg Arg AspCys Ser Lys Val Trp Cys Gly Trp Lys 870 875 880 cct tgt ctg ctg gcc ggccag ccc gag gcc ctg agc gcc cag tgc cca 2786 Pro Cys Leu Leu Ala Gly GlnPro Glu Ala Leu Ser Ala Gln Cys Pro 885 890 895 ctg ggg caa agg tgc ctggag aag gcc cca ggc cag tgt ctg cga cca 2834 Leu Gly Gln Arg Cys Leu GluLys Ala Pro Gly Gln Cys Leu Arg Pro 900 905 910 915 ccc tgt gag gcc tggggg gag tgc ggc gca gaa gag cca ccg agc acc 2882 Pro Cys Glu Ala Trp GlyGlu Cys Gly Ala Glu Glu Pro Pro Ser Thr 920 925 930 ccc tgc ctg cca cgctcc ggc cac ctg gac aat aac tgt gcc cgc ctc 2930 Pro Cys Leu Pro Arg SerGly His Leu Asp Asn Asn Cys Ala Arg Leu 935 940 945 acc ttg cat ttc aaccgt gac cac gtg ccc cag ggc acc acg gtg ggc 2978 Thr Leu His Phe Asn ArgAsp His Val Pro Gln Gly Thr Thr Val Gly 950 955 960 gcc att tgc tcc gggatc cgc tcc ctg cca gcc aca agg gct gtg gca 3026 Ala Ile Cys Ser Gly IleArg Ser Leu Pro Ala Thr Arg Ala Val Ala 965 970 975 cgg gac cgc ctg ctggtg ttg ctt tgc gac cgg gcg tcc tcg ggg gcc 3074 Arg Asp Arg Leu Leu ValLeu Leu Cys Asp Arg Ala Ser Ser Gly Ala 980 985 990 995 agt gcc gtg gaggtg gcc gtg tcc ttc agc cct gcc agg gac ctg cct 3122 Ser Ala Val Glu ValAla Val Ser Phe Ser Pro Ala Arg Asp Leu Pro 1000 1005 1010 gac agc agcctg atc cag ggc gcg gcc cac gcc atc gtg gcc gcc atc 3170 Asp Ser Ser LeuIle Gln Gly Ala Ala His Ala Ile Val Ala Ala Ile 1015 1020 1025 acc cagcgg ggg aac agc tca ctg ctc ctg gct gtc acc gag gtc aag 3218 Thr Gln ArgGly Asn Ser Ser Leu Leu Leu Ala Val Thr Glu Val Lys 1030 1035 1040 gtggag acg gtt gtt acg ggc ggc tct tcc aca ggt ctg ctg gtg cct 3266 Val GluThr Val Val Thr Gly Gly Ser Ser Thr Gly Leu Leu Val Pro 1045 1050 1055gtg ctg tgt ggt gcc ttc agc gtg ctg tgg ctg gcg tgc gtg gtc ctg 3314 ValLeu Cys Gly Ala Phe Ser Val Leu Trp Leu Ala Cys Val Val Leu 1060 10651070 1075 tgc gtg tgg tgg aca cgc aag cgc agg aaa gag cgg gag agg agccgg 3362 Cys Val Trp Trp Thr Arg Lys Arg Arg Lys Glu Arg Glu Arg Ser Arg1080 1085 1090 ctg ccg cgg gag gag agc gcc aac aac cag tgg gcc ccg ctcaac ccc 3410 Leu Pro Arg Glu Glu Ser Ala Asn Asn Gln Trp Ala Pro Leu AsnPro 1095 1100 1105 atc cgc aac ccc atc gag cgg ccg ggg ggc cac aag gacgtg ctc tac 3458 Ile Arg Asn Pro Ile Glu Arg Pro Gly Gly His Lys Asp ValLeu Tyr 1110 1115 1120 cag tgc aag aac ttc acg ccg ccg ccg cgc agg gcggac gag gcg ctg 3506 Gln Cys Lys Asn Phe Thr Pro Pro Pro Arg Arg Ala AspGlu Ala Leu 1125 1130 1135 ccc ggg ccg gcc ggc cac gcg gcc gtc agg gaggat gag gag gac gag 3554 Pro Gly Pro Ala Gly His Ala Ala Val Arg Glu AspGlu Glu Asp Glu 1140 1145 1150 1155 gat ctg ggc cgc ggt gag gag gac tccctg gag gcg gag aag ttc ctc 3602 Asp Leu Gly Arg Gly Glu Glu Asp Ser LeuGlu Ala Glu Lys Phe Leu 1160 1165 1170 tca cac aaa ttc acc aaa gat cctggc cgc tcg ccg ggg agg ccg gcc 3650 Ser His Lys Phe Thr Lys Asp Pro GlyArg Ser Pro Gly Arg Pro Ala 1175 1180 1185 cac tgg gcc tca ggc ccc aaagtg gac aac cgc gcg gtc agg agc atc 3698 His Trp Ala Ser Gly Pro Lys ValAsp Asn Arg Ala Val Arg Ser Ile 1190 1195 1200 aat gag gcc cgc tac gccggc aag gag taggggcggc tgccagctgg 3745 Asn Glu Ala Arg Tyr Ala Gly LysGlu 1205 1210 gccgggaccc agggccctcg gtgggagcca tgccgtctgc cggacccggaggccgaggcc 3805 atgtgcatag tttctttatt ttgtgtaaaa aaaccaccaa aaacaaaaaccaaatgttta 3865 ttttctacgt ttctttaacc ttgtataaat tattcagtaa ctgtcaggctgaaaacaatg 3925 gagtattctc ggaaaaaaaa aaaaaaaaaa 3955 <210> SEQ ID NO 5<211> LENGTH: 1238 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 5 Met Arg Ala Gln Gly Arg Gly Arg Leu Pro Arg Arg Leu Leu LeuLeu -25 -20 -15 Leu Ala Leu Trp Val Gln Ala Ala Arg Pro Met Gly Tyr PheGlu Leu -10 -5 -1 1 5 Gln Leu Ser Ala Leu Arg Asn Val Asn Gly Glu LeuLeu Ser Gly Ala 10 15 20 Cys Cys Asp Gly Asp Gly Arg Thr Thr Arg Ala GlyGly Cys Gly His 25 30 35 Asp Glu Cys Asp Thr Tyr Val Arg Val Cys Leu LysGlu Tyr Gln Ala 40 45 50 Lys Val Thr Pro Thr Gly Pro Cys Ser Tyr Gly HisGly Ala Thr Pro 55 60 65 70 Val Leu Gly Gly Asn Ser Phe Tyr Leu Pro ProAla Gly Ala Ala Gly 75 80 85 Asp Arg Ala Arg Ala Arg Ala Arg Ala Gly GlyAsp Gln Asp Pro Gly 90 95 100 Leu Val Val Ile Pro Phe Gln Phe Ala TrpPro Arg Ser Phe Thr Leu 105 110 115 Ile Val Glu Ala Trp Asp Trp Asp AsnAsp Thr Thr Pro Asn Glu Glu 120 125 130 Leu Leu Ile Glu Arg Val Ser HisAla Gly Met Ile Asn Pro Glu Asp 135 140 145 150 Arg Trp Lys Ser Leu HisPhe Ser Gly His Val Ala His Leu Glu Leu 155 160 165 Gln Ile Arg Val ArgCys Asp Glu Asn Tyr Tyr Ser Ala Thr Cys Asn 170 175 180 Lys Phe Cys ArgPro Arg Asn Asp Phe Phe Gly His Tyr Thr Cys Asp 185 190 195 Gln Tyr GlyAsn Lys Ala Cys Met Asp Gly Trp Met Gly Lys Glu Cys 200 205 210 Lys GluAla Val Cys Lys Gln Gly Cys Asn Leu Leu His Gly Gly Cys 215 220 225 230Thr Val Pro Gly Glu Cys Arg Cys Ser Tyr Gly Trp Gln Gly Arg Phe 235 240245 Cys Asp Glu Cys Val Pro Tyr Pro Gly Cys Val His Gly Ser Cys Val 250255 260 Glu Pro Trp Gln Cys Asn Cys Glu Thr Asn Trp Gly Gly Leu Leu Cys265 270 275 Asp Lys Asp Leu Asn Tyr Cys Gly Ser His His Pro Cys Thr AsnGly 280 285 290 Gly Thr Cys Met Asn Ala Glu Pro Asp Gln Tyr Arg Cys ThrCys Pro 295 300 305 310 Asp Gly Tyr Ser Gly Arg Asn Cys Glu Lys Ala GluHis Ala Cys Thr 315 320 325 Ser Asn Pro Cys Ala Asn Gly Gly Ser Cys HisGlu Val Pro Ser Gly 330 335 340 Phe Glu Cys His Cys Pro Ser Gly Trp SerGly Pro Thr Cys Ala Leu 345 350 355 Asp Ile Asp Glu Cys Ala Ser Asn ProCys Ala Ala Gly Gly Thr Cys 360 365 370 Val Asp Gln Val Asp Gly Phe GluCys Ile Cys Pro Glu Gln Trp Val 375 380 385 390 Gly Ala Thr Cys Gln LeuAsp Ala Asn Glu Cys Glu Gly Lys Pro Cys 395 400 405 Leu Asn Ala Phe SerCys Lys Asn Leu Ile Gly Gly Tyr Tyr Cys Asp 410 415 420 Cys Ile Pro GlyTrp Lys Gly Ile Asn Cys His Ile Asn Val Asn Asp 425 430 435 Cys Arg GlyGln Cys Gln His Gly Gly Thr Cys Lys Asp Leu Val Asn 440 445 450 Gly TyrGln Cys Val Cys Pro Arg Gly Phe Gly Gly Arg His Cys Glu 455 460 465 470Leu Glu Arg Asp Lys Cys Ala Ser Ser Pro Cys His Ser Gly Gly Leu 475 480485 Cys Glu Asp Leu Ala Asp Gly Phe His Cys His Cys Pro Gln Gly Phe 490495 500 Ser Gly Pro Leu Cys Glu Val Asp Val Asp Leu Cys Glu Pro Ser Pro505 510 515 Cys Arg Asn Gly Ala Arg Cys Tyr Asn Leu Glu Gly Asp Tyr TyrCys 520 525 530 Ala Cys Pro Asp Asp Phe Gly Gly Lys Asn Cys Ser Val ProArg Glu 535 540 545 550 Pro Cys Pro Gly Gly Ala Cys Arg Val Ile Asp GlyCys Gly Ser Asp 555 560 565 Ala Gly Pro Gly Met Pro Gly Thr Ala Ala SerGly Val Cys Gly Pro 570 575 580 His Gly Arg Cys Val Ser Gln Pro Gly GlyAsn Phe Ser Cys Ile Cys 585 590 595 Asp Ser Gly Phe Thr Gly Thr Tyr CysHis Glu Asn Ile Asp Asp Cys 600 605 610 Leu Gly Gln Pro Cys Arg Asn GlyGly Thr Cys Ile Asp Glu Val Asp 615 620 625 630 Ala Phe Arg Cys Phe CysPro Ser Gly Trp Glu Gly Glu Leu Cys Asp 635 640 645 Thr Asn Pro Asn AspCys Leu Pro Asp Pro Cys His Ser Arg Gly Arg 650 655 660 Cys Tyr Asp LeuVal Asn Asp Phe Tyr Cys Ala Cys Asp Asp Gly Trp 665 670 675 Lys Gly LysThr Cys His Ser Arg Glu Phe Gln Cys Asp Ala Tyr Thr 680 685 690 Cys SerAsn Gly Gly Thr Cys Tyr Asp Ser Gly Asp Thr Phe Arg Cys 695 700 705 710Ala Trp Pro Pro Gly Trp Lys Gly Ser Thr Cys Ala Val Ala Lys Asn 715 720725 Ser Ser Cys Leu Pro Asn Pro Cys Val Asn Gly Gly Thr Cys Val Gly 730735 740 Ser Gly Ala Ser Phe Ser Cys Ile Cys Arg Asp Gly Trp Glu Gly Arg745 750 755 Thr Cys Thr His Asn Thr Asn Asp Cys Asn Pro Leu Pro Cys TyrAsn 760 765 770 Gly Gly Ile Cys Val Asp Gly Val Asn Trp Phe Arg Cys GluCys Ala 775 780 785 790 Pro Gly Phe Ala Gly Pro Asp Cys Arg Ile Asn IleAsp Glu Cys Gln 795 800 805 Ser Ser Pro Cys Ala Tyr Gly Ala Thr Cys ValAsp Glu Ile Asn Gly 810 815 820 Tyr Arg Cys Ser Cys Pro Pro Gly Arg AlaGly Pro Arg Cys Gln Glu 825 830 835 Val Ile Gly Phe Gly Arg Ser Cys TrpSer Arg Gly Thr Pro Phe Pro 840 845 850 His Gly Ser Ser Trp Val Glu AspCys Asn Ser Cys Arg Cys Leu Asp 855 860 865 870 Gly Arg Arg Asp Cys SerLys Val Trp Cys Gly Trp Lys Pro Cys Leu 875 880 885 Leu Ala Gly Gln ProGlu Ala Leu Ser Ala Gln Cys Pro Leu Gly Gln 890 895 900 Arg Cys Leu GluLys Ala Pro Gly Gln Cys Leu Arg Pro Pro Cys Glu 905 910 915 Ala Trp GlyGlu Cys Gly Ala Glu Glu Pro Pro Ser Thr Pro Cys Leu 920 925 930 Pro ArgSer Gly His Leu Asp Asn Asn Cys Ala Arg Leu Thr Leu His 935 940 945 950Phe Asn Arg Asp His Val Pro Gln Gly Thr Thr Val Gly Ala Ile Cys 955 960965 Ser Gly Ile Arg Ser Leu Pro Ala Thr Arg Ala Val Ala Arg Asp Arg 970975 980 Leu Leu Val Leu Leu Cys Asp Arg Ala Ser Ser Gly Ala Ser Ala Val985 990 995 Glu Val Ala Val Ser Phe Ser Pro Ala Arg Asp Leu Pro Asp SerSer 1000 1005 1010 Leu Ile Gln Gly Ala Ala His Ala Ile Val Ala Ala IleThr Gln Arg 1015 1020 1025 1030 Gly Asn Ser Ser Leu Leu Leu Ala Val ThrGlu Val Lys Val Glu Thr 1035 1040 1045 Val Val Thr Gly Gly Ser Ser ThrGly Leu Leu Val Pro Val Leu Cys 1050 1055 1060 Gly Ala Phe Ser Val LeuTrp Leu Ala Cys Val Val Leu Cys Val Trp 1065 1070 1075 Trp Thr Arg LysArg Arg Lys Glu Arg Glu Arg Ser Arg Leu Pro Arg 1080 1085 1090 Glu GluSer Ala Asn Asn Gln Trp Ala Pro Leu Asn Pro Ile Arg Asn 1095 1100 11051110 Pro Ile Glu Arg Pro Gly Gly His Lys Asp Val Leu Tyr Gln Cys Lys1115 1120 1125 Asn Phe Thr Pro Pro Pro Arg Arg Ala Asp Glu Ala Leu ProGly Pro 1130 1135 1140 Ala Gly His Ala Ala Val Arg Glu Asp Glu Glu AspGlu Asp Leu Gly 1145 1150 1155 Arg Gly Glu Glu Asp Ser Leu Glu Ala GluLys Phe Leu Ser His Lys 1160 1165 1170 Phe Thr Lys Asp Pro Gly Arg SerPro Gly Arg Pro Ala His Trp Ala 1175 1180 1185 1190 Ser Gly Pro Lys ValAsp Asn Arg Ala Val Arg Ser Ile Asn Glu Ala 1195 1200 1205 Arg Tyr AlaGly Lys Glu 1210 <210> SEQ ID NO 6 <211> LENGTH: 4208 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (409)..(4062) <221> NAME/KEY: sig_peptide <222> LOCATION:(409)..(501) <221> NAME/KEY: mat_peptide <222> LOCATION: (502)..(4062)<400> SEQUENCE: 6 ggccggcccg cgagctaggc tggttttttt ttttctcccc tccctcccccctttttccat 60 gcagctgatc taaaagggaa taaaaggctg cgcataatca taataataaaagaaggggag 120 cgcgagagaa ggaaagaaag ccgggaggtg gaagaggagg gggagcgtctcaaagaagcg 180 atcagaataa taaaaggagg ccgggctctt tgccttctgg aacgggccgctcttgaaagg 240 gcttttgaaa agtggtgttg ttttccagtc gtgcatgctc caatcggcggagtatattag 300 agccgggacg cggcggccgc aggggcagcg gcgacggcag caccggcggcagcaccagcg 360 cgaacagcag cggcggcgtc ccgagtgccc gcggcgcgcg gcgcagcg atgcgt tcc 417 Met Arg Ser -30 cca cgg acg cgc ggc cgg tcc ggg cgc ccc ctaagc ctc ctg ctc gcc 465 Pro Arg Thr Arg Gly Arg Ser Gly Arg Pro Leu SerLeu Leu Leu Ala -25 -20 -15 ctg ctc tgt gcc ctg cga gcc aag gtg tgt ggggcc tcg ggt cag ttc 513 Leu Leu Cys Ala Leu Arg Ala Lys Val Cys Gly AlaSer Gly Gln Phe -10 -5 -1 1 gag ttg gag atc ctg tcc atg cag aac gtg aacggg gag ctg cag aac 561 Glu Leu Glu Ile Leu Ser Met Gln Asn Val Asn GlyGlu Leu Gln Asn 5 10 15 20 ggg aac tgc tgc ggc ggc gcc cgg aac ccg ggagac cgc aag tgc acc 609 Gly Asn Cys Cys Gly Gly Ala Arg Asn Pro Gly AspArg Lys Cys Thr 25 30 35 cgc gac gag tct gac aca tac ttc aaa gtg tgc ctcaag gag tat cag 657 Arg Asp Glu Ser Asp Thr Tyr Phe Lys Val Cys Leu LysGlu Tyr Gln 40 45 50 tcc cgc gtc acg gcc ggg ggg ccc tgc agc ttc ggc tcaggg tcc acg 705 Ser Arg Val Thr Ala Gly Gly Pro Cys Ser Phe Gly Ser GlySer Thr 55 60 65 cct gtc atc ggg ggc aac acc ttc aac ctc aag gcc agc cgcggc aac 753 Pro Val Ile Gly Gly Asn Thr Phe Asn Leu Lys Ala Ser Arg GlyAsn 70 75 80 gac cgc aac cgc atc gtg ctg cct ttc agt ttc gcc tgg ccg aggtcc 801 Asp Arg Asn Arg Ile Val Leu Pro Phe Ser Phe Ala Trp Pro Arg Ser85 90 95 100 tat acg ttg ctt gtg gag gcg tgg gat tcc agt aat gac acc gttcaa 849 Tyr Thr Leu Leu Val Glu Ala Trp Asp Ser Ser Asn Asp Thr Val Gln105 110 115 cct gac agt att att gaa aag gct tct cac tcg ggc atg atc aacccc 897 Pro Asp Ser Ile Ile Glu Lys Ala Ser His Ser Gly Met Ile Asn Pro120 125 130 agc cgg cag tgg cag acg ctg aag cag aac acg ggc gtt gcc cacttt 945 Ser Arg Gln Trp Gln Thr Leu Lys Gln Asn Thr Gly Val Ala His Phe135 140 145 gag tat cag atc cgc gtg acc tgt gat gac tac tac tat ggc tttggc 993 Glu Tyr Gln Ile Arg Val Thr Cys Asp Asp Tyr Tyr Tyr Gly Phe Gly150 155 160 tgc aat aag ttc tgc cgc ccc aga gat gac ttc ttt gga cac tatgcc 1041 Cys Asn Lys Phe Cys Arg Pro Arg Asp Asp Phe Phe Gly His Tyr Ala165 170 175 180 tgt gac cag aat ggc aac aaa act tgc atg gaa ggc tgg atgggc ccc 1089 Cys Asp Gln Asn Gly Asn Lys Thr Cys Met Glu Gly Trp Met GlyPro 185 190 195 gaa tgt aac aga gct att tgc cga caa ggc tgc agt cct aagcat ggg 1137 Glu Cys Asn Arg Ala Ile Cys Arg Gln Gly Cys Ser Pro Lys HisGly 200 205 210 tct tgc aaa ctc cca ggt gac tgc agg tgc cag tac ggc tggcaa ggc 1185 Ser Cys Lys Leu Pro Gly Asp Cys Arg Cys Gln Tyr Gly Trp GlnGly 215 220 225 ctg tac tgt gat aag tgc atc cca cac ccg gga tgc gtc cacggc atc 1233 Leu Tyr Cys Asp Lys Cys Ile Pro His Pro Gly Cys Val His GlyIle 230 235 240 tgt aat gag ccc tgg cag tgc ctc tgt gag acc aac tgg ggcggc cag 1281 Cys Asn Glu Pro Trp Gln Cys Leu Cys Glu Thr Asn Trp Gly GlyGln 245 250 255 260 ctc tgt gac aaa gat ctc aat tac tgt ggg act cat cagccg tgt ctc 1329 Leu Cys Asp Lys Asp Leu Asn Tyr Cys Gly Thr His Gln ProCys Leu 265 270 275 aac ggg gga act tgt agc aac aca ggc cct gac aaa tatcag tgt tcc 1377 Asn Gly Gly Thr Cys Ser Asn Thr Gly Pro Asp Lys Tyr GlnCys Ser 280 285 290 tgc cct gag ggg tat tca gga ccc aac tct gaa att gctgag cac gcc 1425 Cys Pro Glu Gly Tyr Ser Gly Pro Asn Ser Glu Ile Ala GluHis Ala 295 300 305 tgc ctc tct gat ccc tgt cac aac aga ggc agc tgt aaggag acc tcc 1473 Cys Leu Ser Asp Pro Cys His Asn Arg Gly Ser Cys Lys GluThr Ser 310 315 320 ctg ggc ttt gag tgt gag tgt tcc cca ggc tgg acc ggcccc aca tgc 1521 Leu Gly Phe Glu Cys Glu Cys Ser Pro Gly Trp Thr Gly ProThr Cys 325 330 335 340 tct aca aac att gat gac tgt tct cct aat aac tgttcc cac ggg ggc 1569 Ser Thr Asn Ile Asp Asp Cys Ser Pro Asn Asn Cys SerHis Gly Gly 345 350 355 acc tgc cag gac ctg gtt aac gga ttt aag tgt gtgtgc ccc cca cag 1617 Thr Cys Gln Asp Leu Val Asn Gly Phe Lys Cys Val CysPro Pro Gln 360 365 370 tgg act ggg aaa acg tgc cag tta gat gca aat gaatgt gag gcc aaa 1665 Trp Thr Gly Lys Thr Cys Gln Leu Asp Ala Asn Glu CysGlu Ala Lys 375 380 385 cct tgt gta aac gcc aaa tcc tgt aag aat ctc attgcc agc tac tac 1713 Pro Cys Val Asn Ala Lys Ser Cys Lys Asn Leu Ile AlaSer Tyr Tyr 390 395 400 tgc gac tgt ctt ccc ggc tgg atg ggt cag aat tgtgac ata aat att 1761 Cys Asp Cys Leu Pro Gly Trp Met Gly Gln Asn Cys AspIle Asn Ile 405 410 415 420 aat gac tgc ctt ggc cag tgt cag aat gac gcctcc tgt cgg gat ttg 1809 Asn Asp Cys Leu Gly Gln Cys Gln Asn Asp Ala SerCys Arg Asp Leu 425 430 435 gtt aat ggt tat cgc tgt atc tgt cca cct ggctat gca ggc gat cac 1857 Val Asn Gly Tyr Arg Cys Ile Cys Pro Pro Gly TyrAla Gly Asp His 440 445 450 tgt gag aga gac atc gat gaa tgt gcc agc aacccc tgt ttg aat ggg 1905 Cys Glu Arg Asp Ile Asp Glu Cys Ala Ser Asn ProCys Leu Asn Gly 455 460 465 ggt cac tgt cag aat gaa atc aac aga ttc cagtgt ctg tgt ccc act 1953 Gly His Cys Gln Asn Glu Ile Asn Arg Phe Gln CysLeu Cys Pro Thr 470 475 480 ggt ttc tct gga aac ctc tgt cag ctg gac atcgat tat tgt gag cct 2001 Gly Phe Ser Gly Asn Leu Cys Gln Leu Asp Ile AspTyr Cys Glu Pro 485 490 495 500 aat ccc tgc cag aac ggt gcc cag tgc tacaac cgt gcc agt gac tat 2049 Asn Pro Cys Gln Asn Gly Ala Gln Cys Tyr AsnArg Ala Ser Asp Tyr 505 510 515 ttc tgc aag tgc ccc gag gac tat gag ggcaag aac tgc tca cac ctg 2097 Phe Cys Lys Cys Pro Glu Asp Tyr Glu Gly LysAsn Cys Ser His Leu 520 525 530 aaa gac cac tgc cgc acg acc ccc tgt gaagtg att gac agc tgc aca 2145 Lys Asp His Cys Arg Thr Thr Pro Cys Glu ValIle Asp Ser Cys Thr 535 540 545 gtg gcc atg gct tcc aac gac aca cct gaaggg gtg cgg tat att tcc 2193 Val Ala Met Ala Ser Asn Asp Thr Pro Glu GlyVal Arg Tyr Ile Ser 550 555 560 tcc aac gtc tgt ggt cct cac ggg aag tgcaag agt cag tcg gga ggc 2241 Ser Asn Val Cys Gly Pro His Gly Lys Cys LysSer Gln Ser Gly Gly 565 570 575 580 aaa ttc acc tgt gac tgt aac aaa ggcttc acg gga aca tac tgc cat 2289 Lys Phe Thr Cys Asp Cys Asn Lys Gly PheThr Gly Thr Tyr Cys His 585 590 595 gaa aat att aat gac tgt gag agc aaccct tgt aga aac ggt ggc act 2337 Glu Asn Ile Asn Asp Cys Glu Ser Asn ProCys Arg Asn Gly Gly Thr 600 605 610 tgc atc gat ggt gtc aac tcc tac aagtgc atc tgt agt gac ggc tgg 2385 Cys Ile Asp Gly Val Asn Ser Tyr Lys CysIle Cys Ser Asp Gly Trp 615 620 625 gag ggg gcc tac tgt gaa acc aat attaat gac tgc agc cag aac ccc 2433 Glu Gly Ala Tyr Cys Glu Thr Asn Ile AsnAsp Cys Ser Gln Asn Pro 630 635 640 tgc cac aat ggg ggc acg tgt cgc gacctg gtc aat gac ttc tac tgt 2481 Cys His Asn Gly Gly Thr Cys Arg Asp LeuVal Asn Asp Phe Tyr Cys 645 650 655 660 gac tgt aaa aat ggg tgg aaa ggaaag acc tgc cac tca cgt gac agt 2529 Asp Cys Lys Asn Gly Trp Lys Gly LysThr Cys His Ser Arg Asp Ser 665 670 675 cag tgt gat gag gcc acg tgc aacaac ggt ggc acc tgc tat gat gag 2577 Gln Cys Asp Glu Ala Thr Cys Asn AsnGly Gly Thr Cys Tyr Asp Glu 680 685 690 ggg gat gct ttt aag tgc atg tgtcct ggc ggc tgg gaa gga aca acc 2625 Gly Asp Ala Phe Lys Cys Met Cys ProGly Gly Trp Glu Gly Thr Thr 695 700 705 tgt aac ata gcc cga aac agt agctgc ctg ccc aac ccc tgc cat aat 2673 Cys Asn Ile Ala Arg Asn Ser Ser CysLeu Pro Asn Pro Cys His Asn 710 715 720 ggg ggc aca tgt gtg gtc aac ggcgag tcc ttt acg tgc gtc tgc aag 2721 Gly Gly Thr Cys Val Val Asn Gly GluSer Phe Thr Cys Val Cys Lys 725 730 735 740 gaa ggc tgg gag ggg ccc atctgt gct cag aat acc aat gac tgc agc 2769 Glu Gly Trp Glu Gly Pro Ile CysAla Gln Asn Thr Asn Asp Cys Ser 745 750 755 cct cat ccc tgt tac aac agcggc acc tgt gtg gat gga gac aac tgg 2817 Pro His Pro Cys Tyr Asn Ser GlyThr Cys Val Asp Gly Asp Asn Trp 760 765 770 tac cgg tgc gaa tgt gcc ccgggt ttt gct ggg ccc gac tgc aga ata 2865 Tyr Arg Cys Glu Cys Ala Pro GlyPhe Ala Gly Pro Asp Cys Arg Ile 775 780 785 aac atc aat gaa tgc cag tcttca cct tgt gcc ttt gga gcg acc tgt 2913 Asn Ile Asn Glu Cys Gln Ser SerPro Cys Ala Phe Gly Ala Thr Cys 790 795 800 gtg gat gag atc aat ggc taccgg tgt gtc tgc cct cca ggg cac agt 2961 Val Asp Glu Ile Asn Gly Tyr ArgCys Val Cys Pro Pro Gly His Ser 805 810 815 820 ggt gcc aag tgc cag gaagtt tca ggg aga cct tgc atc acc atg ggg 3009 Gly Ala Lys Cys Gln Glu ValSer Gly Arg Pro Cys Ile Thr Met Gly 825 830 835 agt gtg ata cca gat ggggcc aaa tgg gat gat gac tgt aat acc tgc 3057 Ser Val Ile Pro Asp Gly AlaLys Trp Asp Asp Asp Cys Asn Thr Cys 840 845 850 cag tgc ctg aat gga cggatc gcc tgc tca aag gtc tgg tgt ggc cct 3105 Gln Cys Leu Asn Gly Arg IleAla Cys Ser Lys Val Trp Cys Gly Pro 855 860 865 cga cct tgc ctg ctc cacaaa ggg cac agc gag tgc ccc agc ggg cag 3153 Arg Pro Cys Leu Leu His LysGly His Ser Glu Cys Pro Ser Gly Gln 870 875 880 agc tgc atc ccc atc ctggac gac cag tgc ttc gtc cac ccc tgc act 3201 Ser Cys Ile Pro Ile Leu AspAsp Gln Cys Phe Val His Pro Cys Thr 885 890 895 900 ggt gtg ggc gag tgtcgg tct tcc agt ctc cag ccg gtg aag aca aag 3249 Gly Val Gly Glu Cys ArgSer Ser Ser Leu Gln Pro Val Lys Thr Lys 905 910 915 tgc acc tct gac tcctat tac cag gat aac tgt gcg aac atc aca ttt 3297 Cys Thr Ser Asp Ser TyrTyr Gln Asp Asn Cys Ala Asn Ile Thr Phe 920 925 930 acc ttt aac aag gagatg atg tca cca ggt ctt act acg gag cac att 3345 Thr Phe Asn Lys Glu MetMet Ser Pro Gly Leu Thr Thr Glu His Ile 935 940 945 tgc agt gaa ttg aggaat ttg aat att ttg aag aat gtt tcc gct gaa 3393 Cys Ser Glu Leu Arg AsnLeu Asn Ile Leu Lys Asn Val Ser Ala Glu 950 955 960 tat tca atc tac atcgct tgc gag cct tcc cct tca gcg aac aat gaa 3441 Tyr Ser Ile Tyr Ile AlaCys Glu Pro Ser Pro Ser Ala Asn Asn Glu 965 970 975 980 ata cat gtg gccatt tct gct gaa gat ata cgg gat gat ggg aac ccg 3489 Ile His Val Ala IleSer Ala Glu Asp Ile Arg Asp Asp Gly Asn Pro 985 990 995 atc aag gaa atcact gac aaa ata atc gat ctt gtt agt aaa cgt gat 3537 Ile Lys Glu Ile ThrAsp Lys Ile Ile Asp Leu Val Ser Lys Arg Asp 1000 1005 1010 gga aac agctcg ctg att gct gcc gtt gca gaa gta aga gtt cag agg 3585 Gly Asn Ser SerLeu Ile Ala Ala Val Ala Glu Val Arg Val Gln Arg 1015 1020 1025 cgg cctctg aag aac aga aca gat ttc ctt gtt ccc ttg ctg agc tct 3633 Arg Pro LeuLys Asn Arg Thr Asp Phe Leu Val Pro Leu Leu Ser Ser 1030 1035 1040 gtctta act gtg gct tgg atc tgt tgc ttg gtg acg gcc ttc tac tgg 3681 Val LeuThr Val Ala Trp Ile Cys Cys Leu Val Thr Ala Phe Tyr Trp 1045 1050 10551060 tgc ctg cgg aag cgg cgg aag ccg ggc agc cac aca cac tca gcc tct3729 Cys Leu Arg Lys Arg Arg Lys Pro Gly Ser His Thr His Ser Ala Ser1065 1070 1075 gag gac aac acc acc aac aac gtg cgg gag cag ctg aac cagatc aaa 3777 Glu Asp Asn Thr Thr Asn Asn Val Arg Glu Gln Leu Asn Gln IleLys 1080 1085 1090 aac ccc att gag aaa cat ggg gcc aac acg gtc ccc atcaag gat tat 3825 Asn Pro Ile Glu Lys His Gly Ala Asn Thr Val Pro Ile LysAsp Tyr 1095 1100 1105 gag aac aag aac tcc aaa atg tct aaa ata agg acacac aat tct gaa 3873 Glu Asn Lys Asn Ser Lys Met Ser Lys Ile Arg Thr HisAsn Ser Glu 1110 1115 1120 gta gaa gag gac gac atg gac aaa cac cag cagaaa gcc cgg ttt gcc 3921 Val Glu Glu Asp Asp Met Asp Lys His Gln Gln LysAla Arg Phe Ala 1125 1130 1135 1140 aag cag ccg gcg tac acg ctg gta gacaga gaa gag aag ccc ccc aac 3969 Lys Gln Pro Ala Tyr Thr Leu Val Asp ArgGlu Glu Lys Pro Pro Asn 1145 1150 1155 ggc acg ccg aca aaa cac cca aactgg aca aac aaa cag gac aac aga 4017 Gly Thr Pro Thr Lys His Pro Asn TrpThr Asn Lys Gln Asp Asn Arg 1160 1165 1170 gac ttg gaa agt gcc cag agctta aac cga atg gag tac atc gta 4062 Asp Leu Glu Ser Ala Gln Ser Leu AsnArg Met Glu Tyr Ile Val 1175 1180 1185 tagcagaccg cgggcactgc cgccgctaggtagagtctga gggcttgtag ttctttaaac 4122 tgtcgtgtca tactcgagtc tgaggccgttgctgacttag aatccctgtg ttaatttaag 4182 ttttgacaag ctggcttaca ctggca 4208<210> SEQ ID NO 7 <211> LENGTH: 1218 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 7 Met Arg Ser Pro Arg Thr Arg Gly Arg SerGly Arg Pro Leu Ser Leu -30 -25 -20 Leu Leu Ala Leu Leu Cys Ala Leu ArgAla Lys Val Cys Gly Ala Ser -15 -10 -5 -1 1 Gly Gln Phe Glu Leu Glu IleLeu Ser Met Gln Asn Val Asn Gly Glu 5 10 15 Leu Gln Asn Gly Asn Cys CysGly Gly Ala Arg Asn Pro Gly Asp Arg 20 25 30 Lys Cys Thr Arg Asp Glu SerAsp Thr Tyr Phe Lys Val Cys Leu Lys 35 40 45 Glu Tyr Gln Ser Arg Val ThrAla Gly Gly Pro Cys Ser Phe Gly Ser 50 55 60 65 Gly Ser Thr Pro Val IleGly Gly Asn Thr Phe Asn Leu Lys Ala Ser 70 75 80 Arg Gly Asn Asp Arg AsnArg Ile Val Leu Pro Phe Ser Phe Ala Trp 85 90 95 Pro Arg Ser Tyr Thr LeuLeu Val Glu Ala Trp Asp Ser Ser Asn Asp 100 105 110 Thr Val Gln Pro AspSer Ile Ile Glu Lys Ala Ser His Ser Gly Met 115 120 125 Ile Asn Pro SerArg Gln Trp Gln Thr Leu Lys Gln Asn Thr Gly Val 130 135 140 145 Ala HisPhe Glu Tyr Gln Ile Arg Val Thr Cys Asp Asp Tyr Tyr Tyr 150 155 160 GlyPhe Gly Cys Asn Lys Phe Cys Arg Pro Arg Asp Asp Phe Phe Gly 165 170 175His Tyr Ala Cys Asp Gln Asn Gly Asn Lys Thr Cys Met Glu Gly Trp 180 185190 Met Gly Pro Glu Cys Asn Arg Ala Ile Cys Arg Gln Gly Cys Ser Pro 195200 205 Lys His Gly Ser Cys Lys Leu Pro Gly Asp Cys Arg Cys Gln Tyr Gly210 215 220 225 Trp Gln Gly Leu Tyr Cys Asp Lys Cys Ile Pro His Pro GlyCys Val 230 235 240 His Gly Ile Cys Asn Glu Pro Trp Gln Cys Leu Cys GluThr Asn Trp 245 250 255 Gly Gly Gln Leu Cys Asp Lys Asp Leu Asn Tyr CysGly Thr His Gln 260 265 270 Pro Cys Leu Asn Gly Gly Thr Cys Ser Asn ThrGly Pro Asp Lys Tyr 275 280 285 Gln Cys Ser Cys Pro Glu Gly Tyr Ser GlyPro Asn Ser Glu Ile Ala 290 295 300 305 Glu His Ala Cys Leu Ser Asp ProCys His Asn Arg Gly Ser Cys Lys 310 315 320 Glu Thr Ser Leu Gly Phe GluCys Glu Cys Ser Pro Gly Trp Thr Gly 325 330 335 Pro Thr Cys Ser Thr AsnIle Asp Asp Cys Ser Pro Asn Asn Cys Ser 340 345 350 His Gly Gly Thr CysGln Asp Leu Val Asn Gly Phe Lys Cys Val Cys 355 360 365 Pro Pro Gln TrpThr Gly Lys Thr Cys Gln Leu Asp Ala Asn Glu Cys 370 375 380 385 Glu AlaLys Pro Cys Val Asn Ala Lys Ser Cys Lys Asn Leu Ile Ala 390 395 400 SerTyr Tyr Cys Asp Cys Leu Pro Gly Trp Met Gly Gln Asn Cys Asp 405 410 415Ile Asn Ile Asn Asp Cys Leu Gly Gln Cys Gln Asn Asp Ala Ser Cys 420 425430 Arg Asp Leu Val Asn Gly Tyr Arg Cys Ile Cys Pro Pro Gly Tyr Ala 435440 445 Gly Asp His Cys Glu Arg Asp Ile Asp Glu Cys Ala Ser Asn Pro Cys450 455 460 465 Leu Asn Gly Gly His Cys Gln Asn Glu Ile Asn Arg Phe GlnCys Leu 470 475 480 Cys Pro Thr Gly Phe Ser Gly Asn Leu Cys Gln Leu AspIle Asp Tyr 485 490 495 Cys Glu Pro Asn Pro Cys Gln Asn Gly Ala Gln CysTyr Asn Arg Ala 500 505 510 Ser Asp Tyr Phe Cys Lys Cys Pro Glu Asp TyrGlu Gly Lys Asn Cys 515 520 525 Ser His Leu Lys Asp His Cys Arg Thr ThrPro Cys Glu Val Ile Asp 530 535 540 545 Ser Cys Thr Val Ala Met Ala SerAsn Asp Thr Pro Glu Gly Val Arg 550 555 560 Tyr Ile Ser Ser Asn Val CysGly Pro His Gly Lys Cys Lys Ser Gln 565 570 575 Ser Gly Gly Lys Phe ThrCys Asp Cys Asn Lys Gly Phe Thr Gly Thr 580 585 590 Tyr Cys His Glu AsnIle Asn Asp Cys Glu Ser Asn Pro Cys Arg Asn 595 600 605 Gly Gly Thr CysIle Asp Gly Val Asn Ser Tyr Lys Cys Ile Cys Ser 610 615 620 625 Asp GlyTrp Glu Gly Ala Tyr Cys Glu Thr Asn Ile Asn Asp Cys Ser 630 635 640 GlnAsn Pro Cys His Asn Gly Gly Thr Cys Arg Asp Leu Val Asn Asp 645 650 655Phe Tyr Cys Asp Cys Lys Asn Gly Trp Lys Gly Lys Thr Cys His Ser 660 665670 Arg Asp Ser Gln Cys Asp Glu Ala Thr Cys Asn Asn Gly Gly Thr Cys 675680 685 Tyr Asp Glu Gly Asp Ala Phe Lys Cys Met Cys Pro Gly Gly Trp Glu690 695 700 705 Gly Thr Thr Cys Asn Ile Ala Arg Asn Ser Ser Cys Leu ProAsn Pro 710 715 720 Cys His Asn Gly Gly Thr Cys Val Val Asn Gly Glu SerPhe Thr Cys 725 730 735 Val Cys Lys Glu Gly Trp Glu Gly Pro Ile Cys AlaGln Asn Thr Asn 740 745 750 Asp Cys Ser Pro His Pro Cys Tyr Asn Ser GlyThr Cys Val Asp Gly 755 760 765 Asp Asn Trp Tyr Arg Cys Glu Cys Ala ProGly Phe Ala Gly Pro Asp 770 775 780 785 Cys Arg Ile Asn Ile Asn Glu CysGln Ser Ser Pro Cys Ala Phe Gly 790 795 800 Ala Thr Cys Val Asp Glu IleAsn Gly Tyr Arg Cys Val Cys Pro Pro 805 810 815 Gly His Ser Gly Ala LysCys Gln Glu Val Ser Gly Arg Pro Cys Ile 820 825 830 Thr Met Gly Ser ValIle Pro Asp Gly Ala Lys Trp Asp Asp Asp Cys 835 840 845 Asn Thr Cys GlnCys Leu Asn Gly Arg Ile Ala Cys Ser Lys Val Trp 850 855 860 865 Cys GlyPro Arg Pro Cys Leu Leu His Lys Gly His Ser Glu Cys Pro 870 875 880 SerGly Gln Ser Cys Ile Pro Ile Leu Asp Asp Gln Cys Phe Val His 885 890 895Pro Cys Thr Gly Val Gly Glu Cys Arg Ser Ser Ser Leu Gln Pro Val 900 905910 Lys Thr Lys Cys Thr Ser Asp Ser Tyr Tyr Gln Asp Asn Cys Ala Asn 915920 925 Ile Thr Phe Thr Phe Asn Lys Glu Met Met Ser Pro Gly Leu Thr Thr930 935 940 945 Glu His Ile Cys Ser Glu Leu Arg Asn Leu Asn Ile Leu LysAsn Val 950 955 960 Ser Ala Glu Tyr Ser Ile Tyr Ile Ala Cys Glu Pro SerPro Ser Ala 965 970 975 Asn Asn Glu Ile His Val Ala Ile Ser Ala Glu AspIle Arg Asp Asp 980 985 990 Gly Asn Pro Ile Lys Glu Ile Thr Asp Lys IleIle Asp Leu Val Ser 995 1000 1005 Lys Arg Asp Gly Asn Ser Ser Leu IleAla Ala Val Ala Glu Val Arg 1010 1015 1020 1025 Val Gln Arg Arg Pro LeuLys Asn Arg Thr Asp Phe Leu Val Pro Leu 1030 1035 1040 Leu Ser Ser ValLeu Thr Val Ala Trp Ile Cys Cys Leu Val Thr Ala 1045 1050 1055 Phe TyrTrp Cys Leu Arg Lys Arg Arg Lys Pro Gly Ser His Thr His 1060 1065 1070Ser Ala Ser Glu Asp Asn Thr Thr Asn Asn Val Arg Glu Gln Leu Asn 10751080 1085 Gln Ile Lys Asn Pro Ile Glu Lys His Gly Ala Asn Thr Val ProIle 1090 1095 1100 1105 Lys Asp Tyr Glu Asn Lys Asn Ser Lys Met Ser LysIle Arg Thr His 1110 1115 1120 Asn Ser Glu Val Glu Glu Asp Asp Met AspLys His Gln Gln Lys Ala 1125 1130 1135 Arg Phe Ala Lys Gln Pro Ala TyrThr Leu Val Asp Arg Glu Glu Lys 1140 1145 1150 Pro Pro Asn Gly Thr ProThr Lys His Pro Asn Trp Thr Asn Lys Gln 1155 1160 1165 Asp Asn Arg AspLeu Glu Ser Ala Gln Ser Leu Asn Arg Met Glu Tyr 1170 1175 1180 1185 IleVal <210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: synthetic DNA <223> OTHER INFORMATION: “n”represents a, t, c, g, other or unknown <400> SEQUENCE: 8 tgcststgyganaccaactg 20 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic DNA <400> SEQUENCE: 9tttatktcrc awktckgwcc 20 <210> SEQ ID NO 10 <211> LENGTH: 180 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 10 gagtgtgcacctggcttcgc ggggcctgac tgccgcatca acatcgacga gtgccagtcc 60 tcgccctgtgcctacggggc cacgtgtgtg gatgagatca acgggtatcg ctgtagctgc 120 ccacccggccgagccggccc ccggtgccag gaagtgatcg ggttcgggag atcctgctgg 180 <210> SEQ IDNO 11 <211> LENGTH: 252 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 11 gcatcaactg ccatatcaac gtcaacgact gtcgcgggcagtgtcagcat gggggcacct 60 gcaaggacct ggtgaacggg taccagtgtg tgtgcccacggggcttcgga ggccggcatt 120 gcgagctgga acgagacaag tgtgccagca gcccctgccacagcggcggc ctctgcgagg 180 acctggccga cggcttccac tgccactgcc cccagggcttctccgggcct ctctgtgagg 240 tggatgtcga cc 252 <210> SEQ ID NO 12 <211>LENGTH: 184 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:12 caacttttcc tgcatctgtg acagtggctt tactggcacc tactgccatg agaacattga 60cgactgcctg ggccagccct gccgcaatgg gggcacatgc atcgatgagg tggacgcctt 120ccgctgcttc tgccccagcg gctgggaggg cgagctctgc gacaccaatc ccaacgactg 180cctt 184 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic DNA <400> SEQUENCE: 13gagtgtgcac ctggcttcgc 20 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic DNA <400>SEQUENCE: 14 ccagcaggat ctcccgaacc 20 <210> SEQ ID NO 15 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: syntheticDNA <400> SEQUENCE: 15 gcatcaactg ccatatcaac 20 <210> SEQ ID NO 16 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic DNA <400> SEQUENCE: 16 ggtcgacatc cacctcacag 20 <210> SEQ IDNO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic DNA <400> SEQUENCE: 17 caacttttcctgcatctgtg 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic DNA <400> SEQUENCE: 18aaggcagtcg ttgggattgg 20 <210> SEQ ID NO 19 <211> LENGTH: 397 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 19 atgaggagctgctgatcgag cgagtgtcgc atgccggcat gatcaacccg gaggaccgct 60 ggaagagcctgcacttcagc ggccacgtgg cgcacctgga gctgcagatc cgcgtgcgct 120 gcgacgagaactactacagc gccacttgca acaagttctg ccggccccgc aacgactttt 180 tcggccactacacctgcgac cagtacggca acaaggcctg catggacggc tggatgggca 240 aggagtgcaaggaagctgtg tgtaaacaag ggtgtaattt gctccacggg ggatgcaccg 300 tgcctggggagtgcaggtgc agctacggct ggcaagggag gttctgcgat gagtgtgtcc 360 cctaccccggctgcgtgcat ggcagttgtg tggagcc 397 <210> SEQ ID NO 20 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: synthetic DNA<400> SEQUENCE: 20 atgaggagct gctgatcgag 20 <210> SEQ ID NO 21 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic DNA <400> SEQUENCE: 21 ggctccacac aactgcccat g 21 <210> SEQ IDNO 22 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic DNA <400> SEQUENCE: 22 tcgcgggggcaatgcgggcg cagggccggg ggcgccttcc ccggcggctg ctgctgctgc 60 <210> SEQ IDNO 23 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic DNA <400> SEQUENCE: 23 cacacgcgcacgtacgtgtc gc 22 <210> SEQ ID NO 24 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic DNA <221>NAME/KEY: CDS <222> LOCATION: (1)..(24) <400> SEQUENCE: 24 gat tat aaagat gat gat gat aaa tga 27 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> SEQID NO 25 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic amino acid <400> SEQUENCE: 25 Asp Tyr LysAsp Asp Asp Asp Lys 1 5 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic DNA <400>SEQUENCE: 26 gctccgggat ccgctccctg 20 <210> SEQ ID NO 27 <211> LENGTH:33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: syntheticDNA <400> SEQUENCE: 27 ctcgagcaac ctgtggaaga gccgcccgta aca 33 <210> SEQID NO 28 <211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: synthetic DNA <400> SEQUENCE: 28 ctcgagtcatttatcatcat catctttata atcacctgtg gaagagccgc ccgtaaca 58 <210> SEQ ID NO29 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: synthetic DNA <400> SEQUENCE: 29 agatctcctg tggaagagccgcccgtaaca a 31 <210> SEQ ID NO 30 <211> LENGTH: 58 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: synthetic DNA <400>SEQUENCE: 30 ctcgagtcat ttatcatcat catctttata atcctccttg ccggcgtagcgggcctca 58 <210> SEQ ID NO 31 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: synthetic DNA <400> SEQUENCE: 31aaggatcccg agggtgtctg ctggaagcca ggctca 36 <210> SEQ ID NO 32 <211>LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:synthetic DNA <400> SEQUENCE: 32 cctctagagt cgcggccgtc gcactcattt acc 33

What is claimed is:
 1. An isolated polypeptide comprising the amino acidsequence of SEQ ID NO.
 1. 2. The polypeptide according to claim 1comprising the amino acid sequence of SEQ ID NO
 2. 3. A compositioncomprising the polypeptide of claim 2 in combination with apharmaceutically acceptable carrier.
 4. A cell culture medium forculturing cells comprising the polypeptide of claim
 2. 5. The cellculture medium according to claim 4 wherein the cells areundifferentiated blood cells.
 6. The polypeptide according to claim 1comprising the amino acid sequence of SEQ ID NO
 3. 7. The polypeptideaccording to claim 1 having differentiation suppressive action againstundifferentiated cells.
 8. The polypeptide according to claim 7 whereinthe undifferentiated cells are other than those of brain and nervoussystem or muscular system stem cells.
 9. The polypeptide according toclaim 7 wherein the undifferentiated cells are undifferentiated bloodcells.
 10. The polypeptide according to claim 1 having suppressiveaction against proliferation of vascular endothelial cells.
 11. Acomposition comprising the polypeptide of claim 1 in combination with apharmaceutically acceptable carrier.
 12. A cell culture medium forculturing cells, comprising the polypeptide of claim
 1. 13. The cellculture medium according to claim 12 wherein the cells to be culturedare undifferentiated blood cells.
 14. An isolated DNA encoding apolypeptide comprising the amino acid sequence of SEQ ID NO.
 1. 15. TheDNA according to claim 14 comprising nucleotides 90-731 of SEQ ID NO. 4.16. An isolated DNA encoding a polypeptide comprising the amino acidsequence of SEQ ID NO.
 2. 17. The DNA according to claim 16 comprisingnucleotides 90-3254 of SEQ ID NO.
 4. 18. An isolated DNA encoding apolypeptide comprising the amino acid sequence of SEQ ID NO.
 3. 19. TheDNA according to claim 18 comprising nucleotides 90-3725 of SEQ ID NO.4.
 20. A recombinant DNA formed by ligating a DNA encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO. 1 to an expressionvetor.
 21. A cell transformed by the recombinant DNA of claim
 14. 22. Aprocess for production of a polypeptide comprising culturing cells ofclaim 21 in a medium and isolating the polypeptide encoded by therecombinant DNA.
 23. A recombinant DNA formed by ligating a DNA encodinga polypeptide comprising the amino acid sequence of SEQ ID NO. 2 to anexpression vector.
 24. A cell transformed by the recombinant DNA ofclaim
 23. 25. A process for production of a polypeptide comprisingculturing cells of claim 24 in a medium and isolating the polypeptideencoded by the recombinant DNA.
 26. A recombinant DNA formed by ligatinga DNA encoding a polypeptide comprising the amino acid sequence of SEQID NO. 3 to an expression vector.
 27. A cell transformed by therecombinant DNA of claim
 16. 28. A process for production of apolypeptide comprising culturing cells of claim 21 in a medium andisolating the polypeptide encoded by the recombinant DNA.